extfieldsofstudy: Biology Medicine

text:
Genome-wide identification and expression analysis of AP2/ERF transcription factors in sugarcane (Saccharum spontaneum L.)

Background APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors play essential roles in plant growth, development, metabolism, and responses to biotic and abiotic stresses. However, few studies concerning AP2/ERF genes in sugarcane which are the most critical sugar and energy crops worldwide. Results A total of 218 AP2/ERF genes were identified in the Saccharum spontaneum genome. Phylogenetic analysis showed that these genes could be divided into four groups, including 43 AP2s, 160 ERFs and Dehydration-responsive element-binding (DREB) factors, 11 ABI3/VPs (RAV), and four Soloist genes. These genes were unevenly distributed on 32 chromosomes. The structural analysis of SsAP2/ERF genes showed that 91 SsAP2/ERFs lacked introns. Sugarcane and sorghum had a collinear relationship between 168 SsAP2/ERF genes and sorghum AP2/ERF genes that reflected their similarity. Multiple cis-regulatory elements (CREs) present in the SsAP2/ERF promoter were related to abiotic stresses, suggesting that SsAP2/ERF activity could contribute to sugarcane adaptation to environmental changes. The tissue-specific analysis showed spatiotemporal expression of SsAP2/ERF in the stems and leaves of sugarcane at different development stages. In ten sugarcane samples, 39 SsAP2/ERFs were not expressed, whereas 58 SsAP2/ERFs were expressed in all samples. Quantitative PCR experiments showed that SsERF52 expression was up-regulated under salt stress, but suppressed under dehydration stress. SsSoloist4 had the most considerable upregulation in response to treatment with the exogenous hormones ABA and GA. Within 3 h of ABA or PEG6000 treatment, SsSoloist4 expression was up-regulated, indicating that this gene could play a role in the responses to ABA and GA-associated dehydration stress. Analysis of AP2/ERF gene expression patterns under different treatments indicated that SsAP2/ERF genes played an essential role in dehydration and salt stress responses of S. spontaneum. Conclusions In this study, a total of 218 members of the AP2 / ERF superfamily were identified in sugarcane, and their genetic structure, evolution characteristics, and expression patterns were studied and analyzed. The results of this study provide a foundation for future analyses to elucidate the importance of AP2/ERF transcription factors in the function and molecular breeding of sugarcane.

Background
Unfavorable environmental conditions have severe impacts on crop yields, and thus strategies to improve crop survival under adverse conditions are needed [1]. Plants respond to environmental stresses via complex regulatory mechanisms that elicit a series of physiological and biochemical responses [2]. Transcription factors play an essential role in converting stress-induced signals into cellular responses. When various abiotic stresses stimulate plants, signaling pathways involving molecules such as abscisic acid and ethylene are activated [3]. This activation is often associated with changes in the expression of transcription factors, which specifically bind to transacting elements in promoter regions at downstream target genes. For cis-acting elements, the regulatory effect is executed through the activation or inhibition of downstream functional genes [4]. In plants, these two main processes are involved in responses to biotic or abiotic stress, which are mediated by various transcription factors. The AP2/EREBP (APETALA2/ethylene response element-binding protein) superfamily comprises a large class of transcription factors in plants. Multiple studies have demonstrated that AP2/ERF transcription factors in plants are essential for stresses responses, and their expression is regulated by plant hormones [5,6]. In response to stresses in plants, AP2/ERF transcription factors' expression is regulated to coordinate growth under stresses [7].
AP2/ERF family members contain the highly conserved AP2/ERF DNA binding domain. Based on sequence similarities and the number of AP2/ERF domains, the AP2/ERF superfamily can be divided into four categories: AP2, ERF (Ethylene-responsive factor), RAV (related to ABI3/VP1), and Soloist [8]. In most cases, the AP2 family contains proteins with two AP2/ ERF domains involved in regulating plant developmental processes [9]. RAV proteins contain two different DNAbinding domains (AP2 and B3), which are regulated by ethylene or brassinosteroid hormones and also are involved in biotic and abiotic stress responses [10,11]. The ERF family is divided into two subfamilies: ERF and DREB (dewater-responsive element binding). Both ERF and DREB contain only one AP2/ERF domain and are critical regulators of plant responses to biotic and abiotic stresses [12]. The Soloist group contains only one AP2 conserved domain and forms a separate group based on its significant structural difference from the other AP2/ ERF family members. However, there are limited evidence that the Soloist group members are positive regulators of SA-mediated plant defense against pathogens [13].
AP2/ERF transcription factors also play a crucial role in abiotic stress responses in plants. For example, OsER-EBP1 and OsEREBP2 modulate the expression of OsRMC, a negative regulator of rice salt stress [20]. Overexpression of maize ZmDBP3 enhances tolerance of transgenic Arabidopsis to drought and cold stresses [21]. In contrast, overexpression of the WIN1 gene from Sorghum confers drought resistance to Arabidopsis by regulating the epidermis [22]. The ERF and DREB families, in particular, contain members that have excellent performance in response to abiotic stress. The DREB protein can specifically bind the A/GCCGAC (DRE/CRT) element related to genes involved in responses to ABA, drought, and low temperature, while ERF subfamily members can interact with the core sequence AGCC GCC (GCC-box) of the ethylene response element (ERE). Such binding to ERE element regulates responses to ethylene and abiotic stresses, and promotes disease resistance [23]. However, many reports suggest that both Arabidopsis ERF and DREB can be combined with DRE/ CRT or ERE elements, indicating that they have a potential role in abiotic [24]. DREBs belong to the ABAindependent signal transduction pathway and are divided into two subclasses: DREB1/CBF and DREB2. DREB1/ CBF genes are thought to be involved mainly in lowtemperature sensation, whereas most DREB2 genes participate in response to water or heat shock stress [25]. However, there is increasing evidence that the stress regulation mediated by the DREB1/CBF and DREB2 genes is species-specific [26]. AP2/ERF transcription factors are also likely to be essential mediators of plant resistance, but few studies concern these genes' activity in sugarcane.
Sugarcane (Saccharum spp.) is the most important crop for sugar and biofuel [27]. Sugarcane provides 75 and 40% of global sugar and ethanol production [27,28]. Damage to sugarcane caused by environmental stresses thus can have substantial economic impacts [29]. Drought stress during sugarcane growth reduces productivity by between 30 and 70%, and minimizes sucrose formation and sucrose recovery by 5% [30]. Genomewide analysis of the presence of AP2/ERF transcription factors in wild sugarcane Saccharum spontaneum species would be necessary for sugarcane resistance research. In this study, we identified members of the AP2/ERF superfamily in the S. spontaneum genome. We also carried out the phylogenetic relationships, gene structure, conserved domains, promoters, chromosomal location distribution, and gene duplication. The effects of AP2/ERF genes on sugarcane adaptation to environmental stresses were analyzed to enhance our understanding of the mechanisms.

Results
Identification and classification of AP2/ERF genes A total of 218 complete AP2/ERF genes were identified in the sugarcane genome database. The identified genes ranged from 416 to 22,786 bp and were predicted to encode proteins with 127-876 amino acids (aa) (Additional files 1 and 2). Based on sequence similarities and the number of conserved AP2 domains (Additional files 3 and 4), the AP2/ERF genes were divided into four families: AP2, ERF, RAV, and Soloist. The AP2 family had 43 genes, of which 36 had two identical conserved AP2 domains, and 7 had only one conserved AP2 domain (SsAP2-37 to SsAP2-43). The ERF family had 160 genes, 59 and 101 were assigned to DREB (SsDREB1 to SsDREB59) and ERF (SsERF1 to SsERF101) subfamily. The RAV family comprised of 11 members (SsRAV1 to SsRAV11) with conserved AP2 and B3 domains. Another four genes (SsSoloist1 to SsSoloist4) with one or two conserved AP2 domain had less similarity to ERF or AP2. Instead, these four genes had higher homology to Arabidopsis At4g13040, and thus were classified into the Soloist. Because no reliable naming method was defined in previous research on the AP2/ ERF family, our naming convention was based on the grouping of 218 sequences and their chromosomal positions. The total number of AP2/ERF superfamily candidate genes in sugarcane was higher than that in the Arabidopsis (147) and rice (181) [8]. However, the number of ERF family members was similar but slightly higher (122 and 145 for Arabidopsis and rice, respectively).

Phylogenetic analysis
To analyze the evolutionary relationship of the SsAP2/ ERF genes, we constructed phylogenetic trees based on the amino acid sequences encoded by these genes (Fig. 1). The phylogenetic tree clustered all of the SsAP2/ERF genes into four main branches: AP2, ERF, RAV, and Soloist. According to the classification criteria in Arabidopsis and rice [8], the ERF family was divided into two subfamilies: the DREB subfamily (59 members, divided into groups I, II, III, and IV, containing 19,11,19, and 10 members, respectively) and the ERF subfamily (101 members divided into groups V, VI, VII, VIII, IX, X, and XIV containing 12, 11, 12, 18, 24, 16 and 8 members, respectively) ( Fig. 2). Notably, most species lacked a XIV group, although Os08g41030 in rice had a conserved domain similar to these eight sugarcane members and could be classified into the XIV group. Arabidopsis had no genes that were consistent with the XIV group. Thus, an additional examination is needed to determine whether functional differentiation occurred and its significance.

Gene structure and conserved motif analysis
To characterize the structural diversity of SsAP2/ERF genes, we analyzed the number of introns and exons and the distribution of conserved domains in the coding sequence of a single SsAP2/ERF gene (Additional file 5: Figure S6). Through gene structure analysis, differences during the AP2, ERF, and RAV families could be observed, and the results from the conserved domain analysis were consistent with those of previous studies. The number of introns among the different family genes varied markedly (Fig. 3), and 42% of AP2/ERF genes had no intron. Most of DREB genes had no intron except for 18 genes, such as SsDREB16, SsDREB20, SsDREB49, SsDREB50, which the number of introns ranged from 1 to 24. A total of 40 ERF genes had no intron. Unlike the ERF genes, 42 AP2 genes had the number of introns ranging from 2 to 9. For the 11 RAV genes, SsRAV9 had one intron, SsRAV5 had six introns, while the remaining nine RAV genes had on intron. The number of introns of the four Soloist genes was between 6 and 9. Interestingly, the genes clustered into the same branch on the phylogenetic tree had similar exon-intron structure. For example, DREB group II genes had no intron except the gene SsDREB33, which had only one intron.
We used MEME to investigate SsAP2/ERF gene diversity further and predicted the presence of 25 conserved motifs in the AP2/ERF family (Additional file 6 and Figure 3). Motif-1 and -2 were present in all SsAP2/ERF protein sequences; Both motif-3 and -4 were available in most members of the AP2, ERF, DREB, and RAV subfamily; Motif-6, − 7, − 8, − 20, and − 22 were found only in the AP2 family; Motif-10, − 12 and − 15 appeared in group I; Motif-13 appeared in group XIV, and Motif-9 and -11 were unique to the RAV family. We also found that Motif-1, − 2, and − 7 existed in the AP2 conserved domain, whereas Motif-9, − 11, and − 16 were located in the B3 domain (Fig. 3). As expected, most close relatives among subfamily members had similar motif compositions, suggesting that AP2/ERF proteins within the same subfamily were functionally identical.

Chromosome distribution
The 218 identified SsAP2/ERF genes were mapped to 32 chromosomes, and the distribution across the chromosomes varied widely (Fig. 4). Chr2A had the most genes (12), whereas Chr6C and Chr8C had only three genes (Additional file 7). At least one of the 59 SsDREB and 101 SsERF genes could be found on each of the 32 chromosomes, and the four Soloist genes were distributed on four homologous chromosomes, Chr4A, Chr4B, Chr4C, and Chr4D. Members of the SsRAV family were present only on chromosomes 3 and 7. Surprisingly, 50% of the SsAP2/ERF genes localized at one of the four rearrangement chromosomes (SsChr02, SsChr05, SsChr06 and SsChr07) and nine SsAP2/ERF genes were present in the rearranged regions, including SsChr5 (Sb05S) 57.6-89.

Gene duplication analysis
Multiple studies indicated that gene families evolved due to genome-wide duplication, segmental duplication or tandem duplication, and gene diversification occurred after these duplication events [32]. To explore SsAP2/ ERF genes evolution, we studied tandem and segmental duplication events of these genes using chromosomal information for S. spontaneum (Additional file 8). A total of eight pairs of tandem duplication genes were detected, of which two pairs were RAV genes, and six ones were ERF genes (Fig. 4). Three genes, SsRAV4, SsRAV5, and SsRAV6, were two tandem duplication gene pairs located on the same chromosome and adjacent to each other. Besides, 70 pairs of 103 SsAP2/ERF segmental duplication genes were detected (Fig. 5). Among these, 53 occurred between alleles, and 17 were between nonalleles. There were four pairs of SsAP2/ERF segmental duplication gene pairs distributed on Chr4 and Chr5. The distribution might be due to the segments of both Chr4 and Chr5 chromosomes from the ancestral A4 chromosome [31].
The time for the divergence of the SsAP2/ERF genes was estimated to have undergone tandem and segmental duplication based on Ks values (Additional file 8). The divergence time for the eight SsAP2/ERF tandem duplication pairs ranged from 14.2 to 104.2 million years ago (mya), illustrating that these SsAP2/ERFs arose from recent gene duplication events in S. spontaneum. Sixtyfour segmental duplication pairs appeared earlier, based on a divergence time that ranged from 4.9 to 89.9 mya, whereas the other four segmental duplication pairs were ancient, diverging between 164.7 and 212.1 mya.
We calculated Ka/Ks values for SsAP2/ERF genes in tandem and segmental duplications to detect which selection type promoted AP2/ERF gene family evolution (Additional file 8). The Ka/Ks ratio of tandem duplication gene pairs among AP2/ERF genes ranged from 0.17 to 1.24, with an average of 0.57. Tandem duplication gene pairs having a Ka/Ks ratio < 1 accounted for 89% of the genes tested. The Ka/Ks ratio for segmental duplication gene pairs ranged from 0 to 2.6, with an average of 0.62, and 92% of pairs had Ka/Ks < 1. These results indicated that repetitive SsAP2/ERF genes were under intense purification selection pressure, and the duplication-producing gene had enormously evolved and maintained its functional stability. Meanwhile, nine replicate gene pairs had Ka/Ks > 1, indicating that they were subject to positive selection after duplication differentiation.

Evolutionary analysis of SsAP2/ERFs and other plant AP2/ ERFs
A syntenic map of five representative species was constructed to examine the evolutionary origin of the AP2/ ERF genes in sugarcane ( Fig. 6 and Additional file 9). A total of 168 SsAP2/ERF genes were synonymous with genes in Sorghum, followed by rice (151), wheat (145),  (19). Orthologous gene pairs were distributed across all chromosomes of S. spontaneum. Some SsAP2/ERF genes were associated with at least three pairs of corresponding genes (particularly AP2/ERF genes in sugarcane and wheat), suggesting that these genes played an essential role in SsAP2/ERF superfamily evolution. Interestingly, some collinear gene pairs (90 SsAP2/ERF genes) were available in monocotyledonous plants (sugarcane and rice/sorghum/wheat/ maize), but absent in the dicotyledonous ones (sugarcane and Arabidopsis).

Analysis of putative cis-regulatory elements (CREs)
CRE is a non-coding DNA sequence that regulates transcription in a defined temporal/spatial expression pattern. CREs are essential for understanding expression differences and mutations. We used PlantCare to identify putative CREs of 2000 bp located on SsAP2/ERF genes ( Fig. 7 and Additional file 10). These CREs were classified according to their function, and the number of CRE in each sequence was determined. Many CREs responded to different hormone inducers, such as abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), gibberellin (GA), and auxin. Among them, ABA and MeJA response elements were the most common. There are also CREs genes related to various stress stimuli (such as a wound, defense, and stress, anoxic, anaerobic, and low-temperature). A total of 182 SsAP2/ERF genes contained the abscisic acid response element, 126 genes with the gibberellin response element, and 192 genes with the MeJA response element. Another 108 SsAP2 / ERF genes contained low-temperature response elements, while 155 genes had light responsiveness elements. Each family had ten types of CREs, including light responsiveness, endosperm expression, anoxic specific inducibility, auxin, gibberellin, abscisic acid, MeJA, and salicylic acid responsiveness, MYBHv1, and MYB binding site. DREB genes had the most CREs species, among which alpha-amylase promoters and root-specific CREs only existed in DREB subfamily genes. The CREs Fig. 3 Phylogenetic relationships, gene structure, and architecture of conserved protein motifs in AP2/ERF genes from sugarcane. The phylogenetic tree was constructed based on the full-length sequences of sugarcane AP2/ERF proteins using MEGA7 software-exon and intron structure of sugarcane AP2/ERF genes. Yellow boxes indicated untranslated 5 -and 3 -regions; green boxes indicated exons; gray lines indicated introns. The AP2 domains were highlighted by red boxes and the B3 domain by light green boxes. The motifs, numbers 1-25, were displayed in different colored boxes. The sequence information for each motif was provided in Additional file 6. The protein and gene length can be estimated using the scale at the bottom of Soloist family genes were the least, mainly hormone and stress response. The five families of genes had similar CREs types, but the number of copies of each CRE was different. In some AP2/ERF genes, the promoter region contained cis-acting elements for various transcription factors, such as MYB, MYC, ERE and DRE elements.

Expression pattern of AP2/ERF genes during sugarcane development
To understand specific spatiotemporal expression patterns of SsAP2/ERF genes, we analyzed the identified genes' expression profiles in ten different tissues and at different developmental stages using publicly available gene expression data (Additional files 11 and 12). Among the SsAP2/ERF genes examined, 39 were not expressed in all tissue samples, 58 were expressed in all ten tissue types (FPKM > 0), and 27 were constitutively expressed (FPKM > 2). Total 19 genes in DREB group I was all expressed in at least one sample, among which SsDREB17, SsDREB27, and SsDREB28 were highly expressed in all ones, indicating that these group I members were likely essential functional genes. The expression levels of 26 SsAP2/ERF genes increased during leaf maturation, suggesting that they might play an important role in leaf growth and development. The expression of SsDREB38, SsERF91, SsERF98, SsERF39, and SsERF99 in stems was much higher than that in leaves in the three developmental stages. Among them, four ERF genes belonged to ERF group VII, indicating that they played an essential role in cane stems. Expressions of 11 genes, including SsDREB56, SsDREB48, SsERF15, and SsRAV3, SsSoloist1, and SsSoloist2, were higher in the three stages of leaf development than those in the stems, indicating that these genes played an important role in leaf development. The expression of ten genes, including SsDREB55, SsERF62, and SsERF54 in different tissues in the pre-mature and mature stages, was higher than that seen in various tissues in the seedling stage. Among these genes, many were in the ERF subfamily, indicating that they played an essential role in the mature sugarcane stage. To further confirm whether SsAP2/ERF gene expression was affected by different abiotic stresses and hormone treatments, we examined 12 genes from five families. We analyzed their expression patterns following different treatments (Fig. 8 and Additional file 13). NaCl, PEG6000, and ABA treatment could induce the expression of all 12 genes. After 3 h of treatment, compared with the control group simultaneously, the up-regulation multiple was the highest among all treatment time points. Different treatments had varying degrees of influence on gene expression. Compared with the control group, all the genes were induced by NaCl, except that SsSoloist4 down-regulated expression at 72 h of NaCl treatment. The up-regulation multiple was significantly higher than the other three treatments. Some genes had opposite expression patterns under different treatments. For example, SsERF52 was down-regulated at 1, 6, and 12 h after PEG6000 treatment. Although SsERF52 was up-regulated 2.15 times at 3 h, its up-regulation was significantly lower than that of other genes at 3 h. Meanwhile, the expression of SsERF52 was induced by salt stress at six treatment times. The up-regulated multiple was 15.21 times at 3 h, which was significantly higher than other genes, indicating that the response of SsERF 52 to salt stress and dehydration stress was different. The other four genes, SsAP2-25, SsDREB25, SsRAV11, and SsRAV4, had no significant up-down multiple changes at all time points after GA treatment but were induced by the other three treatments.

Discussion
As one of the largest gene families in plants, the AP2/ ERF family plays a vital role in multiple physiological and biochemical processes by regulating the expression of genes that participate in response to various stress conditions in Arabidopsis, rice [8], maize [33], poplar [34], and grapes [17]. Still, there is limited information concerning the regulation and structure of these genes in sugarcane. In this study, we examined whole-genome data for the wild sugarcane species S. spontaneum to Genome-wide analysis of sugarcane identified 218 SsAP2/ERF genes, which was higher than that in rice (164) [8], wheat (117) [35], and Arabidopsis (145) [8], but fewer than that for maize (292) [33]. The genome size varies in different plants, rice (466 Mb), wheat (4.94 Gb), Arabidopsis (125 Mb), maize (2.3 Gb), and S. spontaneum (3.36 Gb), indicating that the number of AP2/ERF superfamily members is relatively stable and does not have an absolute correlation with genome size. However, since AP85-441 (1n = 4x = 32) used to sequence the S. spontaneum genome was haploid and produced from the culture of the octoploid SES208, the number of AP2/ERF genes in octoploid S. spontaneum could be over 218 [31]. Gene duplication plays a vital role in gene family amplification to generates gene clusters or hotspots via tandem repetitive and segmental duplication to produce homologous genes that expand the total number of genes. Segmental duplication events found in 104 SsAP2/ERF genes also validated this possibility. S. spontaneum has undergone two Whole Genome Duplication (WGD) events overtime in which its homologous chromosomes underwent duplication from one to two and then to four [31]. SsAP2/ERF gene repeats likely occurred during these two WGDs. We also found evidence that purification selection was the main driving force behind the SsAP2/ERF gene family's evolution. By evaluating the gene structure of AP2/ERF TF, all SsAP2 and SsSoloist genes had introns, whereas 50.6 and 83.3% of SsERF/DREB and SsRAV genes, respectively, had no introns. Loss of introns in genes after segmental duplication occurred more rapidly than intron acquisition [36]. Also, some studies showed that the number and distribution of introns were related to plant evolution [37], such that introns might have been lost from ERF and RAV family genes during the evolution of higher plants. From Syntenic analysis of AP2/ERF was carried out to discover the evolutionary relationship of the SsAP2/ERF genes in five monocotyledonous plants (O. sativa, S. bicolor, T. aestivum, Z. mays, S. spontaneum) and one dicotyledonous plant (A. thaliana). In this analysis, SsAP2/ ERF genes had higher homology with AP2/ERF genes from the four kinds of grass and less homology with Arabidopsis. More homologous genes between sugarcane and wheat are likely due to the larger genome size and gene number in wheat [38]. By analyzing the homology of SsAP2/ERF and S. bicolor AP2/ERF genes, we found that 168 SsAP2/ERF genes likely existed before the number of S. spontaneum chromosomes was reduced from 10 to 8. Also, 63-70% of identified SsAP2/ ERF genes were homologous in sorghum (168), rice (147), maize (139), and wheat (145). This finding indicated that the AP2/ERF family in different plants might have evolved from a common ancestor. Due to the plasticity of the AP2/ERF genes and individual family members' specificity, members of this family were likely to be important targets for genetic engineering and breeding to improve crops [39]. The gene expression patterns are essential for the prediction of gene function prediction. Analysis of tissue-and growth stage-specific expression showed that SsAP2/ ERFs were widely expressed in sugarcane at the seedling stage and leaves during early maturity and the mature stage and in different stem segments, indicating that these genes played essential roles in sugarcane growth and development. DREB family genes in group I was highly expressed in leaves at all development stages and in three stem segments, suggesting that these genes might be involved across the entire development process of sugarcane leaves and stems. The expression levels of 29 SsAP2/ERF genes in stems during the seedling stage were lower than those in the early maturity stage and mature stage. Among them, 28 were members of the DREB and ERF family, which might be related to sugar accumulation in the sugarcane stem and consistent with previous studies for other plants. For example, compared with wild rice, transgenic rice carrying the ERF protein TSRF1 had 30-60% increases in proline and soluble sugar content that could enhance the osmotic tolerance of rice [40].
Earlier studies of AP2/ERF family genes in other plants emphasized their role in response to hormonal and Fig. 8 Expression profiles of 12 selected SsAP2/ERF genes in response to NaCl stress treatments. The 25 s-RNA gene was used as the internal control and to normalize expression data. Relative transcript abundance was normalized relative to S mock (1-h untreated control group) treatment. Error bars represented the standard deviation of the mean. Different lowercase letters indicated significant differences among mean values (one-way ANOVA with Ducan's multiple range test; P < 0.05). The results were based on three replicates in three independent experiments abiotic stresses. OsEREBP1 attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice [41]. Induction of the AP2/ERF transcription factor CRL5 promoted crown root initiation by inhibiting cytokinin signaling [42]. In the present study, the expression patterns of 12 SsAP2/ERF genes in response to NaCl, PEG6000, ABA, and GA treatments suggested that these genes had essential roles in abiotic stress and hormone responses sugarcane. qRT-PCR verification revealed that the expressions of these 12 SsAP2/ERF genes distributed across the five subfamilies were significantly induced by NaCl treatment, similar to other species' findings, including the increase in expression of the AP2/ERF family gene CAP2 in chickpeas and soybeans exposed to salt stress [43]. GmERF7 expression increased salt tolerance in tomato [44], whereas the pepper pathogen-induced transcription factor RAV 1 played a vital role in bacterial salt stress tolerance [45]. Here we showed that the expression of SsERF52 was up-regulated by up to 15-fold after NaCl treatment. This gene had the highest homology with the wheat salt-responsive gene, TaERF4 [46]. Like TaERF4 in wheat, SsERF52 expression was not induced by exogenous abscisic acid (ABA). Overexpression of the Arabidopsis AP2/ERF gene HARD Y improved drought tolerance by reducing the transpiration of transgenic Trifolium alexandrinum L [47]. Drought stress induced expression of the DERB OsAP21 in rice [48], and here we found that expression of 12 SsAP2/ERF genes was induced at 3 h after PEG6000 treatment. The expression level of SsERF52 after PEG6000 treatment was opposite that after NaCl treatment, with inhibition of gene expression at several time points. Multiple lines of evidence indicated that genes expressed during dehydration and salt stress responses in plants partially overlapped. For example, transgenic plants that overexpressed Arabidopsis OsMYB3R-2 have enhanced cold tolerance, drought tolerance, and salt tolerance [49]. Increasing amounts of evidence showed that AP2/ERF genes were regulated by hormones such as ABA and GA, and thus these genes played an essential role in hormone signaling networks [50]. In the present study, promoter analysis indicated that most SsAP2/ERF genes' promoter regions contained multiple cis-acting elements related to ABA responses. qRT-PCR analysis of the 12 selected SsAP2/ERF genes showed that, except for SsERF52, expressions of these genes were induced by ABA. AP2 / ERF genes played an active role in ABAmediated stress response, a critical hormone that regulated abiotic stress responses (including drought, salinity, cold, and heat stress) [50]. For example, ANT [51] in Arabidopsis was induced by ABA to up-regulate genes containing DRE and ABRE elements. ANT is homologous to SsAP2-21, indicating that SsAP2/ERF genes might regulate abiotic stress through ABA-dependent pathways. After exogenous GA treatment, the expression levels of SsRAV4, SsRAV11, SsDREB25, and SsAP2-25 were not affected, but GA induced other genes' expression. Our research indicated that some SsAP2/ERF genes were differentially expressed under various abiotic stresses and hormone treatments compared with the control group, suggesting that this gene family was essential for environmental adaptation. In particular, the expression patterns of some SsAP2/ERF genes and their homologs varied. For example, the SsSoloist4 gene expression was induced to varying degrees by the four stress treatments. These expression patterns differed from those for its Arabidopsis homolog At4g13040, an active regulator of disease defenses, and induced by SA treatment. However, the expression of this gene was inhibited by low temperature and salt stress. Given the antagonistic roles of SA and ABA in plant defense, At4g13040 expression may have opposing effects in response to ABA and SA treatment. Compared with the control group, SsSoloist4 expression was up-regulated after 3 h of ABA treatment, indicating that this gene's regulation mechanism likely differed from that for At4g13040 under abiotic stress.
Therefore, it could be seen from the above experiments that 12 SsAP2 / ERF genes were induced and expressed by NaCl and PEG6000 in varying degrees. There were many CREs related to ABA response in the promoter region of these genes, such as ABRE, and more than one copy, which meaned that their fast-induced expression on exogenous ABA treatment might also be mediated by themselves. A total of 12 AP2 / ERF genes were positively regulated by ABA, which further confirmed the result. Among the 12 AP2 / ERF genes, only SsAP2-21 had no CRE related to GA response. However, after GA treatment, the expression of 12 AP2 / ERF genes had no significant correlation with the number of TATC-box box, showing that gene expression was a complex biological process, regulated by many factors. The results also showed that the number of CREs were related to abiotic stress. Still, there was no significant correlation between CREs and 12 SsAP2/ERF genes' gene expression under dehydration and salt stress.
AP2 / ERF gene is regulated by ethylene and brassinosteroid when the plant exposed to salt, low temperature, and flooding stress [52]. AP2 / ERF, especially the ERF subfamily, plays a role as the primary downstream regulator of the ETH signaling pathway [52,53]. For example, in Arabidopsis, EIN3 activate ERF1, ESE, and their downstream stress-related genes, promoting salt tolerance. ETH signal transduction is a necessary condition for plant salt tolerance [54]. However, ethylene's regulation to stress through AP2 / ERF is very complex, which needs further study. BRs also plays a vital role in the abiotic stress response of plants [55]. Recent studies have shown that AP2 / ERF gene is involved in regulating BRs on drought stress and plant growth [56]. BRs signal negatively regulates DREB gene TINY through the phosphorylation of BIN2, and TINY positively regulates drought response and inhibits BRs mediated plant growth through the antagonistic effect of TINY-BES1. These findings provide useful insight to study the relationship between SsAP2 / ERF and BRs in the future. Plants will inevitably suffer from oxidative stress when subjected to biotic and abiotic stresses [57]. The change of oxidation balance might be complex and hurt plants. AP2 / ERF gene is involved in the regulation of oxidative stress response in plants [58]. Oxidative stress strongly induced the ERF gene SICRF1 in tomato [59]. Cytokinin response factor 6 (CRF6) in Arabidopsis played a negative role in oxidative stress response, indicating that the cytokinin mediated the oxidative stress response regulated by AP2 / ERF gene [60]. However, the mechanism of plant response to abiotic stress, including oxidative stress, is still unclear. Sugarcane response to oxidative stress-mediated by SsAP2 / ERF genes is needed for further research in the future. Our results could provide a foundation for identifying novel candidate AP2/ERF genes as targets for genetic engineering of novel sugarcane germplasm that will produce plants with enhanced tolerance of abiotic stresses.

Conclusions
This study comprehensively analyzed the supergene family of sugarcane AP2 / ERF. The 218 identified AP2 / ERF superfamily genes were classified in detail. Their evolutionary characteristics, expression patterns in different sugarcane tissues and growth stages, and their response to abiotic stress and hormones were studied. These results provided valuable resources to understand better the biological role of the sugarcane AP2 / ERF genes.

Identification and classification of members AP2/ERF superfamily genes in sugarcane
The genome sequences and the sequence information of sugarcane were downloaded from the S. spontaneum AP85-441 genome (http://www.life.illinois.edu/ming/ downloads/Spontaneum_genome/) [31]. The protein sequences of AP2/ERF superfamily genes in rice and Arabidopsis were collected from the NCBI (https://www.ncbi. nlm.nih.gov/). These proteins were used as query sequences in the local BLAST program (Basic Local Alignment Search Tool) to find members of AP2/ERF superfamily genes of the sugarcane genome with the following parameters: expected values ≤1E-5. All BLAST hits were checked and searched for conserved AP2 domains online using the Search Pfam feature of the Pfam (https:// pfam.xfam.org/) website under default parameters. In addition, the results of Pfam were verified again using the NCBI CDD tool (https://www.ncbi.nlm.nih.gov/Structure/ bwrpsb/bwrpsb.cgi/) and the cutoff set to 0.01.
Phylogenetic analysis of sugarcane AP2/ERF genes Multiple alignments of candidate AP2/ERF genes were performed to explore the phylogenetic relationship of sugarcane AP2/ERF genes using ClustalW [61] with default parameters. The results were used to construct phylogenetic trees by the neighbor-joining method and were then visualized using MEGA 6.0 software [62]. The phylogenetic trees were generated using complete protein sequences with the following parameters: pair-wise deletion, Poisson correction, and 1000 bootstrap replicate.

Gene structure and conserved motif analyses
Conserved motifs of AP2/ERF proteins were identified using the online tool Multiple Em for Motif Elicitation (MEME) version 5.0.5 [63] (http://meme-suite.org/tools/meme/) with the following parameters: (1) the number of occurrences of a single motif distributed among the sequences within the model was set to zero or one per sequence; (2) the maximum number of motifs found was set as 25; (3) the optimum motif width was set to ≥6 and ≤ 50; and (4) motifs with a matched E-value should be below 0.05. Gene structure was investigated using GSDS 2.0 (http://gsds.cbi.pku.edu.cn/) [64]. We used TBtools software [65] to integrate phylogenetic trees, conserved motifs, and gene structure results.

Chromosomal distribution and duplication analysis of AP2/ERF superfamily genes
The chromosomal distribution information of the identified genes was searched against the reference sugarcane genome database, and the results obtained were visualized using TBtools software. Analysis of gene Duplication events using Multiple collinear scanning toolkits (MCScanX) [66]. The syntenic relationship between the SsAP2/ERF genes and AP2/ERF genes from selected plants was determined by using Dual Synteny Plotter software (https://github.com/CJ-Chen/TBtools/). The putative duplication events were detected for the AP2/ ERF genes. Tandem duplication was identified as two proteins with a greater than 40% similarity and separated by four or fewer gene loci; others were identified as segmental duplications, separated by more than five genes. Non-synonymous (ka) and synonymous (ks) substitution of each duplicated AP2/ERF genes were calculated using KaKs_Calculator 2.0 [67]. The divergence time (T) was calculated by T = Ks/ (2× 6.1 × 10 − 9 ) × 10 − 6 Mya [68]. These results were visualized using TBtools.

Analysis of cis-acting elements of AP2/ERF superfamily genes
Two thousand bp upstream of the transcriptional start site of each SsAP2/ERF gene was selected to inspect potential CREs.

Transcriptome data source and bioinformatic analysis
Sugarcane tissue-specific expression data included leaves and stems at the seedling stage of 35 days and the early maturity stage of 9 months and the maturity stage of 12 months (Additional file 14). All SsAP2 / ERF FPKMs (Segmentals Per Kilobase of transcript per Million segmental mapped) was used to make heat maps and cluster analysis through TBtools.

Plant materials
Sugarcane variety ROC22 is from Guangxi University, Guangxi, China. All the materials were cultured in Murashige and Skoog's medium containing 3% sucrose and 0.7% agar until seedling stage [69]. The seedlings were then transplanted to the greenhouse with light intensity of 100 μ mol / m-2S-1, light cycle of 16:8-h (light / dark), temperature of 28°C, and treated with hormone at 5-7 leaf stage and simulated abiotic stresses. Hormonal treatment was sprayed with abscisic acid (ABA, 100 μM) and gibberellin (GA, 100 μM). Abiotic stress treatment was irrigated with NaCl (250 mM) to simulate salt stress, and PEG6000 (20%) was used to simulate dehydration stress. All the young leaves were taken for samples with the time points are 1, 3, 6, 12, 24, and 72 h. The collected samples were quickly placed in liquid nitrogen and stored in a − 80°C refrigerator for subsequent RNA extraction.

Quantitative real-time PCR (qRT-PCR) analysis
Each qRT-PCR reaction mixture (20 μL) comprised of 10 μL Taq polymerase (SYBR Premix Ex TaqII; Takara), 2 μL of each forward and reverse primers (2 μM), 2 μL of cDNA, and 6 μL of water. The RNA expression level was normalized to the level of 25S-rRNA expression. The amplification was conducted in LightCycler 96 Real-Time PCR System (Roche Lightcyler® 480). A standard thermal profile for SYBR Premix was as followed: cDNA synthesis at 37°C for 15 min and enzyme inactivation at 85°C for 5 s. qPCR conditions were: initial denaturation 95°C for 30s, denaturation 95°C for 5 s, annealing and extension 60°C for 30s. Transcripts expression levels were calculated with the 2 −ΔΔCt method, as previously mentioned in Livak and Schmittgen [70]. Primers used for this analysis are mentioned in Additional file 15.

Data statistics
All data were analyzed for variance using IBM SPSS Statistics. Statistical methods were used to compare the significance levels of LSD (least significant difference test, LSD) at 0.05. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files (from Additional file 1 to Additional file 15). The genome sequences and the sequence information of sugarcane were downloaded from the S. spontaneum AP85-441 genome (http://www.life.illinois.edu/ming/downloads/Spontaneum_ genome/).

Ethics approval and consent to participate
The sugarcane materials used in the experiment were supplied by the sugarcane clonal germplasm repository of Guangxi University. These plant materials are widely used worldwide, and no permits were required for the collection of plant samples. This article did not contain any studies with human participants or animals and did not involve any endangered or protected species.

Consent for publication
Not applicable.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Gene Identification for Bacteriocin Production by Lactic Acid Bacteria Isolated from Selected Fermented Foods

Bacteriocin genes are biosynthetic genes which encodes proteins involved in bacteriocin regulation, self-immunity, transport and modification. This research was aimed at identifying the gene for the synthesis of bacteriocin. Four strains of lactic acid bacteria previously isolated from fermented foods (Nono (N2), Ogi (O3), Dawadawa (D1 and D3) and Wara (W3) were identified using molecular technique and used to produce bacteriocin. The bacteriocin activity was assayed against some test bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella sp and methicillin resistant Staphylococcus aureus) using agar well diffusion method and the bacteriocin genes were identified using BAGEL3. The LAB identified were Lactobacillus fermentum O3, Leuconostoc mesenteroides N2, Weissella cibaria D1 and 2 strains of Lactobacillus plantarum D3 and W3. The entire identified LAB was able to produce bacteriocin. The antimicrobial activity showed varied inhibitory effects of the bacteriocins on the test bacteria. Bacteriocin from isolate O3 showed the highest inhibition zone 16mm on S. aureus. The identified gene for these bacteriocins were plnJK gene for Lactobacillus plantarum str WCFS1 and strain LZ95 (W3 and D3), entA gene for Lactobacillus fermentum str 3872 (O3) and ppnC7 gene for Leuconostoc mesenteroides str SRA3 (N2) with the interaction of other peptides were responsible for bacteriocin production. Original Research Article Abdulkarim et al.; AJBGMB, 3(4): 1-12, 2020; Article no.AJBGMB.55785 2

INTRODUCTION
During fermentation, LAB utilise Carbohydrate substrates available in the fermentation system and produce organic acids, chiefly lactic acid, as a fraction of their metabolites. They produce various substances essential for the development of flavor, aroma, color and texture of fermented foods. Those substances are organic acids, diacetyl, exopolysaccharides, Lalanine, and acetaldehyde. These acids not merely play an important role in the taste and aroma of the product but also lower the product's pH, which is one of the means to ensure quality and safety. It has been reported that several LAB can produce bacteriocins which are active against closely related species [1].
The conventional factors elaborated by LAB have been identified as "peptides", generally known as "bacteriocins". Lactic acid bacteria play a vital role in food fermentation process. Raw foods such as milk, fruits, vegetables or meat are frequently preserved by lactic acid fermentation [2]. In such food products, LAB has the capability to carry out fermentive activities, which may result in dynamic inhibition of pathogenic bacteria. Lactic acid bacteria bacteriocins can work via different mechanisms to exert an antimicrobial effect, but the cell envelope is generally the target.
Bacteriocins are proteins or complex proteins biologically active with antimicrobial action against other bacteria, principally closely related species. Bacteriocins produced by LAB have received considerable attention during recent years for their possible use as biopreservative in foods, with a resultant reduction in the use of chemical preservatives [3]. The bacteria that produce antimicrobial peptides are immune to their own bacteriocins due to the synthesis of specific immunity proteins. According to John and Lennox [4], bacteriocins can be used in three different ways in fermented foods: in situ production by the addition of a bacteriocinogenic lactic culture, as a co-culture and by the direct addition of the bacteriocin.
A typical bacteriocin contains a toxin (bacteriocin) gene, an immunity gene (which confers resistance to the aforementioned toxin) and a lysis gene, which encodes a protein that aids in toxin release from the producing cell [5].
Identification of genes that are functionally similar but have limited or no sequence homology has been a problem, hence the detection of open reading frames ORFs. This is particularly the case for bacteriocins, a very diverse group of antimicrobial peptides produced by bacteria and usually encoded by small, poorly conserved ORFs. ORFs surrounding bacteriocin genes are often biosynthetic genes. This information is used by BAGEL, a web based software to locate putative structural bacteriocin genes [6]. BAGEL scans the bacterial genome for putative bacteriocin open reading frames and also analyses surrounding ORFs to search for possible biosynthetic genes, immunity genes and transporters.

Bacteriocin Genetics
Bacteriocins can be encoded on chromosomes, plasmids, and other transposable elements. The Gram-positive bacteriocins are more complex genetically, with genes that encode posttranslational modification of the toxin. The genetic organization varies between the Grampositive bacteriocin classes as well -such as the requirement of two peptides for the full activation of Class II bacteriocins. An example is the nisin gene cluster which includes 11 genes (nisABTCIPRKFEG) encoding functions such as synthesis of the nisin precursor (nisA), regulation of nisin biosynthesis (nisRK), the processing and translocation of nisin (nisBCTP), and immunity (nisIFEG) [7].

The Structural Prebacteriocin Gene
The structural gene encodes a prebacteriocin, called a precursor or prepeptide. These prepeptides contain an N-terminal leader sequence and a C-terminal propeptide which is cleaved from the N-terminal leader sequence to form a mature, antimicrobial peptide. All Class II bacteriocins were produced as precursors with an N-terminal extention. Most of the leader peptides differ from typical signal secretion peptides that direct polypeptides into secdependent secretion pathways.
The function of leader peptides appears to be the prevention of biological activity of the bacteriocin while still in the producer cell, and to provide a recognition signal for the ABC transporter. Leader peptides may prevent activity of prebacteriocins by increasing the solubility of prebacteriocins in water, causing the peptides to partition into the aqueous phase rather than into the membrane. Leader peptides may also interact with mature peptides and thus reduce their affinity for membranes [8]. The N-terminal leader peptides of Class IIa bacteriocins are referred to as double-glycine leader peptides. These peptides have two glycine residues at the C-terminus before the cleavage site. Other consensus elements include conserved hydrophobic and hydrophilic regions. The minimum length of the leader peptide of nonlanthionine bacteriocins appears to be 14 amino acids, while the length of the mature bacteriocins identified to date varied from 30 to more than 100 residues.

The Immunity Gene
The immunity gene encodes a protein that protects the producer organism from its own mature bacteriocin [8]. A close genetic proximity exists between immunity genes and bacteriocin structural and processing genes. In the case of Class II bacteriocins, one gene generally encodes for the immunity protein. The mechanisms involved in immunity is poorly understood, but it has been suggested that interaction between the immunity protein and another protein, perhaps a receptor, located at the cytoplasmic side of the cell membrane of the producer, protects it against the action of the bacteriocin [9,10]. Potential immunity proteins have been identified next to or downstream from, all bacteriocin structural genes studied. Immunity genes not directly associated with the bacteriocin cluster have also been identified. Variation in the presence and expression of these genes may account for the large variation in sensitivity displayed by lactic acid bacteria towards bacteriocins. Immunity proteins range in sizes from 51 to 150 amino acids. While significant homology exists among the structural genes of the Listeria active bacteriocins, this trend does not occur with immunity genes, although some resemblances do occur [8].

The Transporter Gene
The bacteriocin ABC transporters have a dual function, facilitating both the removal of the leader peptide from its substrate and the transport of the substrate across the cytoplasmic membrane. Bacteriocin ABC-transporters contain three domains on the same polypeptide, consisting of a cytoplasmic N-terminal proteolytic domain, a hydrophobic integral membrane domain, and a cytoplasmic C-terminal ATPbinding domain. Two polypeptides appear to be required for the bacteriocin ABC transporter to be functional.
A unique feature of bacteriocin ABC transporters is that they carry an N-terminal extension of approximately 150 amino acids, the proteolytic domain, which appears to be involved in the processing of the bacteriocins. Two conserved motifs, the cysteine motif (QX4D/ ECX2AX3MX4Y/FGx4I/L) and the histidine motif (HY/FY/VVX10I/LXDP) have been identified in the proteolytic domain and appear to be necessary for translocation. The processing site is part of the transporter, which indicates that the processes of cleavage and translocation are integrated, and that the leader peptide serves as a recognition signal for the transmembrane transport process of the bacteriocin [8].

The Accessory Protein
Several studies have indicated the presence of an additional gene within bacteriocin operons, called the accessory protein (also accessory factor), that is required for the ABC-transporter dependent translocation process. These additional factors have been identified in several Gram-negative systems to be needed when the secreted product is destined for immediate release into the extracellular medium [8]. It is hypothesized that the accessory factor is anchored in the inner membrane and spans the periplasm, probably connecting the inner and outer membranes to facilitate the export of products through both membranes of Gramnegative bacteria. In Gram-positive bacteria, the function of the accessory factor is unclear, since the secreted product only needs to cross one membrane.
The aim of the research is to improve understanding of the mechanisms involved in bacteriocin regulation, processing, translocation and immunity this should facilitate attempts to optimize bacteriocin production and may further open the way to directed in vitro modifications in their antibacterial spectra. Since techniques now available for the site directed mutagenesis of bacteriocin structural genes and with the help of genomics and proteomics, the prospect of constructing new families of designed peptides with improved antimicrobial activity or improved stability and specificity characteristics has become a real possibility.

Bacterial Strains
The lactic acid bacteria strains used in this study were isolated from fermented foods (nono, ogi, wara and dawadawa) and were identified by using molecular techniques according to the method described by Smith et al. [11].

Isolation of Lactic Acid Bacteria (LAB)
Solid samples (dawadawa and wara) were homogenized in disinfected grinder (CombinMax600, China) Using sterilized spatula, 25 g of dawadawa, wara, ogi and 25 ml of nono sample were stocked in 225 ml of peptone water to prepare stock solution. 1 ml of the stock was used for serial dilution in peptone water in test tubes to ten folds dilution [12]. To prevent the growth of yeasts, the media was supplemented with 100 mgL -1 of cycloheximide (Ali, 2011). The MRS agar plates were then incubated aerobically and anaerobically using the Gas Pack system at 37°C for 48 h. Discrete Colonies were randomly selected, purify and subsequently stored at 37°C for further identification [13].

Isolation of Test Bacteria
The test organisms were obtained from the laboratory of Department of Microbiology, Kaduna State University and confirmed using biochemical tests and molecular techniques. Three (3) differential media were used; Mannitol Salt Agar (MSA), Eosin Methylene Blue (EMB) and Salmonella and Shigella agar (SSA). The manufacturer's instruction was used as a template in preparing the required quantity of the media. The media was allowed to cool to 45°C. 20 mLs of the molten agar was aseptically poured onto sterile petri dishes and covered to aid uniformity on plate and allowed to solidify on disinfected bench prior to use. Distinct and well isolated colonies were subcultured and the pure isolates were subjected to biochemical tests [14].

Bacteriocin Assays
The MRS broth was seeded with 5% inoculums of overnight LAB culture and maintained anaerobically at 30°C for 48 hours. After incubation, the broth was centrifuged at 5000rpm for 10 minutes at 4°C and the cells separated out. The cell free supernatant was adjusted to pH 4.0 using 1N NaOH and was used as crude bacteriocin [15].

Partial Purification of the Bacteriocins Produced by LAB
The bacteriocin produced were filtered and precipitated by using 80% ammonium sulphate and the system was held overnight at 4°C. The precipitates were recovered by centrifugation at 15,000 rpm for 30 minutes at 4°C and the pellets were dissolved in 5 ml of 0.05 M potassium phosphate buffer and were designated as crude bacteriocin [16].

Antagonistic Effect of the Bacteriocins on Selected Bacteria Isolates
To determine the bacteriocin activity, well assay procedure of Udhayashree et al. [2], 20 mLs of Muller-Hinton agar was poured into steriled Petridishes and was allowed to solidify for 30 minutes. Five colonies of the test bacteria were inoculated on the agar plates and four wells of 6.0 mm in diameter were aseptically bored using a sterile cock borer on each agar plate. Aliquots of 50µl of the bacteriocin inoculum were poured in the agar wells in Petri dishes seeded with the bio assay strain (indicator microorganism): Staphylococcus aureus, Escherichia coli, Klebsiella spp and methicillin resistant Staphylococcus aureus. The plates were then incubated at 37°C for 24 hours. Effects of the bacteriocins were assessed by measuring the diameters of zone of inhibition in millimetre (mm) around the wells.

Genomic Extraction
The bacterial DNA was extracted and identified using the method by Smith et al. [11].

Identification of the Bacteriocin Gene
The bacterial genome sequence was used as input to detect the gene responsible for bacteriocin production using bagel software (a bacteriocin genome mining tool). BAGEL uses DNA nucleotide sequences in FASTA format as input; multiple sequence entries per file allowed. These DNA sequences are analyzed in parallel using two different approaches, one based on finding genes commonly found in the context of bacteriocin or RiPP genes, the other based on finding the gene itself.The degree of novelty in the bacteriocins identified was determined by BAGEL3 and BLASTP searches for each putative bacteriocin peptide against those identified in the BAGEL screen. The genomic sequences were identified using the NCBI database and the reference sequences generated.

Identification of Lactic Acid Bacteria
The lactic acid bacteria were identified as Weissella cibaria CBA3612, Lactobacillus planterum LZ95 which were isolated from dawadawa, Leuconostoc mesenteroides SRA3 isolated from nono, Lactobacillus fermentum 3872 isolated from ogi and Lactobacillus plantarum WCFS1 from wara.

Bacteriocin Assay
All the lactic acid bacteria identified produced bacteriocin. The bacteriocin produced had varying inhibitory activity against the indicator bacteria, as shown in Table 1

Identified Gene for Bacteriocin Production
The Lactobacillus plantarum WCFS1 genome contains a six gene, 5941 bp Plantaricin cluster ( Table 2). Containing genes predicted to encode a plantaricin structural protein. Immediately upstream are bacteriocin-related genes, including a putative LanM-like lantibiotic modification protein, a putative LanT-like lantibiotic transport protein, ATP-binding and permease protein and some plantaricin immunity protein as shown in (Plate 2).  Lactobacillus plantarum LZ95 genome also contains six precursor bacteriocin gene, 5941 bp plantaricin cluster (Table 3). The area of interest contained genes predicted to encode plantaricin similar to stain WCFS1 genome as shown in (Plate 3).

Plate 1. Amplified 16S rRNA gene of the PCR products
Lactobacillus fermentum 3872 genome contained a putative enterolysin structural protein as shown in (Table 4), enterolysin A is a high molecular weight bacteriocin (42499) together with other uncharacterized protein, membrane peptidase and transposase for insertion element (Plate 4).
Leuconostoc mesenteroides C7 genome contained a putative leucocin structural gene (Table 5). This genome contains an 8 gene 2065 bp cluster. Gene PpnC7 is predicted to encode the structural protein Leucocin K.

DISCUSSION
The production of the bacteriocins were achieved in mixed population, E. coli and Klebsiella pneumonia were added to the broth to achieve a competitive environment. Similar observations by Ogunbonwa et al. [17] also noted that bacteriocins are often produced under stress conditions, such as nutrient limitation and overpopulation. The prevalence of bacteriocin positive strains in the food samples analysed as shown are much higher than those reported by other findings. Sezer and Güven [18] Screened 12,700 LAB isolates from milk and meat products and found only 35 exhibited bacteriocin production. Also, Salasiah et al. [19] screened 3000 colonies of LAB isolated from traditional fermented foods and only one colony was found to produce inhibitory zone. The high incidence of bacteriocin producing LAB in these foods suggests that they may represent an abundant source of potentially useful bacteria.  [20] in raw and malted cereals.
Six genes (Plantaricin K, N, A, E, F and J) for precursor bacteriocin and a transporter gene for bacteriocin ABC transporter was detected in both the Lactobacillus plantarum sequences Plasmid sequence analysis of Lactobacillus plantarum LZ95 (D3) revealed six open reading frames (ORFs) containing one replication protein (ORF00023), three hypothetical proteins (ORF00022, ORF00034 and ORF00048 ORF5), one conserved hypothetical protein (ORF00046) and one mobilization protein (ORF00050). The predicted protein from ORF00023 (putative replication protein) consisted of fifty five (55) amino acid residues, with a weight of 5,941 Da. Also, it is thermostable, stable at a pH of 5.0 to 8 and has a broad inhibition spectrum against Gram positive and Gram negative bacteria.
The gene for Lactobacillus plantarum WCFS1 and Lactobacillus plantarum LZ95 exhibits antimicrobial activity that was subsequently found by six peptides with fifty five (55) amino acid residue and molecular weight of (5941 DA). The gene for this bacteriocin was identified as plnJk and the bacteriocin produced was plantaricin. Wang et al.
[21] isolated Lactobacillus plantarum with similar gene cluster but differ in their molecular weight and hydrophobicity. Gene plnK of these isolates (W3 and D3) were 100% identical to the Lactobacillus plantarum under the accession number WP-003641972, isolated by Nissen-Meyer et al. [22]. Lactobacillus fermentum produced enterolysin bacteriocin and the gene was identified as enlA gene with 404 amino acid residue and 42499 DA molecular weight. Similar report by Nilsen et al.
[23] identified Enterolysin A isolated from Enterococcus faecalis which was 56% positive, 41% identical and had 38% similar proteins. Another report by Altermann et al. [24] identified gene LBA1207 with similar properties. Isolate N2 produced leucocin K bacteriocin and gene ppnC7 with 52 amino acid was identified.
Sequencing of the entire locus of Lactobacillus plantarum WCFS1 showed it to comprise 62 ORF, while Lactobacillus plantarum LZ95 was 65 ORF, then Leuconostoc mesenteroides had 7 ORF and 37 ORF for Lactobacillus fermentum. Sequence analysis and BLAST homologies indicated these are likely to be involved in lantibiotic biosynthesis, regulation, immunity and N-terminal peptide. Weissella cibaria (D1) gene of interest was not identified by the database as it does not have strong homology (partially sequenced) to previously identified peptides. It was also reported that the GenBank database on Weissella genes is too short to predict or identify their specific functions. From that research, five of six ORFs in pKW2124 were not predicted properly, probably due to low GenBank information on Weissella genes [25].
Bacteriocin genes identified belong to class II (a) and (b) bacteriocins due to the presence of double glysine motifs detected by BAGEL. An interesting structural feature of all Class IIb bacteriocins is the presence of GxxxG motifs (where x represents any residue) and GxxxG type motifs [conformed by A (AxxxA) or S (SxxxS)] that facilitate helix-helix interactions and promote the oligomerization of transmembrane helical peptides or membrane protein domains as reported by several authors [26,27]. Although E-760 shares this Class IIb uniqueness, it lacks the second characteristic peptide of this category; since in order to achieve an optimal antibacterial activity, the presence of both peptides is required in approximately equal amounts.

CONCLUSION
From the result, it was observed that for the full activation of bacteriocins in Gram positive lactic acid bacteria the interaction of other genes; biosynthesis, regulation, immunity and transport may be required. As also stated by Bakkal et al. [5] who reported that Class II bacteriocins require two peptides for their full activation.


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The lipid profile of rats (Rattus norvegicus L.) induced by high fat ration after exposed to ethanolic neem (Azadirazchta indica) leaf extract

The objective of this study was to analyze the effect of neem leaf ethanol extract as an herbal antihyperlipidemia agent on white rats induced by high fat ration. The white rat used was male Wistar strain with 2 months of age and average body weight of approximately 200 grams. The rats were divided into 6 groups of 4, namely: Negative control (P0) was given commercial ration, positive control (P1) was given high fat ration and duck egg yolk per oral 2.5 ml / g BW, P2 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 g BW + 8 mg / g BW simvastatin in 1 ml of distilled water, P3 was given high-fat ration and duck egg yolk orally 2.5 ml / 200 g BW + 75 mg / g BW ethanol extract of neem leaf in 1 ml of distilled water, P4 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 g BW + 100 mg / g BW ethanol extract of neem leaf in 1 ml of distilled water, and P5 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 g BW + 125 mg / g BW ethanol extract of neem leaf in 1 ml of distilled water. The variables observed were total cholesterol, HDL, LDL and TG levels. Data were analyzed using ANOVA followed by Least Significant Difference (LSD) test with 95% confidence level using SPSS 10.0 software. The results showed that administration of the ethanol extract of neem leaf (A. indica) can raise levels of HDL, lowering levels of LDL cholesterol and TG in blood serum of white rats (Rattus norvegicus L.).

High-fat ration is a food that contains high concentrations of fat. The intake of fat into the human body in large amounts can increase total blood cholesterol and blood LDL [8]. An increase in cholesterol levels due to consumption of high amounts of fat occurs because in the metabolism of fat, the fat consumed in part will be converted to cholesterol. Fat derived from local synthesis and food, will be transported to the liver. Fat derived from local synthesis is released and transported to the liver in the form of free fatty acids, while fat from food is transported in the form of chylomicrons [9]. Sherwood (2001) states that arterial disease can occur with elevated levels of LDL and VLDL cholesterol in the blood (hypercholesterolemia) [10]. According to Linder (1992)  saturated fat foods in high amounts continuously is a major factor that can increase cholesterol and triglyceride levels in plasma which causes hyperlipidemia [11].
Disease in arteria can occur with increased levels of LDL and VLDL cholesterol in the blood (hypercholesterolemia). This increase in cholesterol levels can occur when there is a disturbance in the formation of cholesterol in the liver or small intestine [10]. High levels of LDL cholesterol will trigger the accumulation of cholesterol in blood vessel cells, which causes the emergence of atherosclerosis and the formation of plaque in the walls of blood vessels [12].
Nevertheless, according to Barter et al. (2007) HDL cholesterol levels and / or the ratio between total cholesterol and HDL cholesterol are strong predictors for the incidence of cardiovascular disease. The higher the HDL cholesterol level in relation to the total cholesterol level, the smaller the risk of cardiovascular disease. It was further said that HDL levels were risk factors for cardiovascular disease that were stronger than LDL levels [13].
According to Nicholson & Hajjar (1998), atherosclerosis is a multifactorial disease, which involves various factors, including: genetic factors, such as: hypercholesterolemia, hypertension, diabetes mellitus and obesity, and environmental factors, including smoking and stress. At present, most people still recognize atherosclerosis as a degenerative disease that develops slowly and is considered a disease that only attacks elderly individuals. Classic factors, which accompany it include hypercholesterolemia, hypertriglyceridemia or hypertension. But research that runs continuously from year to year proves, that atherosclerosis is a disease that is very complicated, exceeding pre-existing assumptions [14].
Azadirachta indica at a dose of 500mg/kg BW can reduce cholesterol levels in diabetic-induced mice [15]. One of the potentials of various compound contents of neem leaves is to reduce cholesterol levels which can trigger heart and blood vessel disease [16]. Repeated administration of A. indica leaf ethanol extract in diabetic rats can prevent an increase in total cholesterol (TC), triglyceride (TG), Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) compared to diabetic rats that are not given neem leaf extract , while High Density Lipoprotein (HDL) cholesterol levels increased significantly [17].
Base on that fact, this study was performed to find out the effect of ethanolic Neem leaf extract on rats serum which characterized by lipid profile i.e. cholesterol, LDL, HDL and TG. The outcome of this study is offering the advantages of Neem leaf extract as an effective herb in antihyperlipidemia.

Preparation of the ethanolic leaf extract of Neem
The neem leaf were collected from Diponegoro University campuss area. Then the leaf was rinsed and dried at 40-50 o C for ten days. The leaf ethanolic extract were made by maseration method using 70% ethanol as explained by Sitasiwi et al. [18]. The extracts were kept in 4 o C in a dark closed bottle until antihyperlipidemia testing.

Making high fat rations
High-fat ration consists of commercial ration and reused cooking oil. Used cooking oil used in this study was obtained from one liter packaged cooking oil used to fry tofu weighing 450 g for 10 minutes at a temperature of 150-165 o C with deep fat frying techniques for nine times the frying pan [19,20].

Laboratory animals
Twenty four male Rattus norvegicus L. rats with 2 months of age and ± 200g in body weight were used as testing animals. The rats were obtained from the Department of Biology Laboratory of Semarang State University, Indonesia. They were acclimatized for seven days in the laboratory condition before being used for experiments. The animals were handled in a well-controlled room, with the temperature of 26 ± 1°C. The rations and water were given ad libitum.

Experimental design and treatment of animals procedures
Research conducted at the Laboratory of Biological Structure and Function of Animals Department of Biology, Faculty of Science and Mathematics University of Diponegoro, in May and July 2019. Highfat ration is given every morning for 45 days as much as ± 30 g, then given drinking water every day along with 75 ml of ration. Giving duck egg yolk is given once every two days (morning), while simvastatin and ethanol extract of neem leaf are given every evening orally for 44 days by syringe injection berkanul tip. The tools used are 24 individual cages equipped with drinking and rationing, measuring cups, digital scales, gives a chance to set. This study uses a completely randomized design with 6 treatments and 4 replicates ie: Negative control (P0) was given commercial ration, positive control (P1) was given high fat ration and duck egg yolk per oral 2.5 ml / g BW, P2 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 gBW + 8 mg / g BW simvastatin in 1 ml of distilled water, P3 given high-fat ration and duck egg yolk orally 2.5 ml / 200 g BW + 75 mg / g BW ethanol extract of neem leaf (A. indica) in 1 ml of distilled water, P4 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 g BW + 100 mg / g BW ethanol extract of neem leaf (A. indica) in 1 ml of distilled water, and P5 was given high fat ration and duck egg yolk per oral 2.5 ml / 200 g BW + 125 mg / g BW ethanol extract of neem leaf (A. indica) in 1 ml of distilled water.

Statistical analysis
The cholesterol, HDL, LDL and TG were expressed as the mean ± standard deviation, each with four replicates. The collected data were analyzed by One-way Analysis of Variance (ANOVA) based on completely randomized design (CRD) at the level of 95% (α = 0.05) and continued by Least Significant Difference Test (LSDT). P-values differences at p<0.05 were considered significant. SPSS was applied to analysed the data.

Results and discussion
The result shows that the ethanolic Neem leaf extract interferes the profile lipid of testing animals (Table 1.). The cholesterol, HDL, LDL and TG of treated animals shows a significant difference (p<0.05) with the group was given high fat ration. Description: Numbers with different superscripts in the same row show the real difference among the treatments. Negative control (P0) was given commercial ration, positive control (P1) was given high fat ration and duck egg yolk per oral 2.5 ml / g BW, P2 was given high fat ration and duck egg yolk per oral 2.5 ml/200 g BW + 8 mg / g BW simvastatin in 1 ml of distilled water, P3 was given high-fat ration and duck egg yolk orally 2. Results of the analysis of the levels of LDL in the treatment giving 75, 100, 125 mg/g BW ethanol extract of neem leaf showed significant differences to the rats were given high fat ration. Results of the analysis of the levels of HDL in the treatment giving 75, 100, 125 mg/g BW ethanol extract of neem leaf showed significant differences to the rats were given high fat ration. Results of the analysis of the levels of TG in the treatment giving 75, 100, 125 mg/g BW ethanol extract of neem leaf showed significant differences to the rats were given high fat ration. Results of the analysis as shown in table 1.
The results showed that the relationship with total cholesterol LDL is directly proportional. This happens because the 45% is in the form of LDL cholesterol [18]. That is if your total cholesterol LDL down then also fell. This happens because the inhibition or disruption of the process of absorption of cholesterol in the small intestine and increasing bile acid excretion in the feces. Bile acids are end products of metabolism of cholesterol. With the high excretion of bile acids, the more cholesterol is converted into bile acids to emulsification of fat, so that the serum total and LDL cholesterol decreased. Mechanism of increased levels of HDL because of the influence of ethanol extract of neem leaf likely caused by the binding of bile acids by metabolites (contained in ethanol extract of neem leaf) in the small intestine that causes increased excretion of bile acid fecal, resulting in decreased absorption of fat and cholesterol, it causes cholesterol in the liver low so cholesterol to produce bile acids less. This condition stimulates the synthesis of HDL in the liver to meet the shortage of cholesterol. As a result, lower serum LDL than HDL serum. Along with a decrease in serum cholesterol levels in the blood of the results of the analysis of HDL levels in treatment giving ethanol extract of neem leaf showed significant differences to the rats were given high fat ration.
Diseases of the arteries can occur with elevated levels of LDL and TG in the blood (hypercholesterolemia). The increase in cholesterol levels can occur when there is an interruption formation of cholesterol in the liver or intestine [10]. High LDL levels will trigger the accumulation of cholesterol in the blood vessel cells, which led to the emergence of atherosclerosis and plaque formation in the blood vessel walls 12 . Atherosclerosis is associated with increased LDL. Effect of elevated levels of LDL will be followed by the accumulation of cholesterol esters in macrophages which then referred to as a macrophage foam cells. High LDL levels lead to high levels of LDL intimal. Furthermore, intimal will oxidize LDL and attracts monocytes from the blood circulation as well as phenotypic change into macrophages. Increased oxidized LDL in the arterial wall is accompanied by the formation of foam cells, will develop into a plate of fat [21].

Conclusions
The results showed that administration of the ethanol extract of neem leaf (A. indica) can raise levels of HDL, lowering levels of LDL cholesterol and TG in blood serum of white rats (Rattus norvegicus). It concluded that ethanol extract of neem leaf (A. indica) could potentially be used as a supplement for the prevention of risk to vascular disease and coronary heart disease.