Transcriptome Analysis And Selecting Of Virulence-Associated Genes And SRNA Prediction For Fish Source Streptococcus Agalactiae

Streptococcus agalactiae is a gram-positive bacteria, also known as Group B streptococcus, one of the main pathogens of leading to tilapia streptococcus disease in southern China. In recent years, tilapia streptococcus disease occur frequently, aquaculture industry has suffered huge economic losses in China, therefore, prevention and research of Streptococcus agalactiae are imminent. Studies have shown that, virulence of S.agalactiae is regulated by the temperature, it can cause outbreak and epidemic of tilapia streptococcus disease when the water temperature exceeds 35℃.In order to research and selecting virulence-associated genes of fish source S.agalactiae, S.agalactiae ZQ0910 was cultured in different temperatures(25℃ and 37℃)as the research object, the bacteria was cultured to logarithmic growth phaseat in 25℃and37℃, respectively, then, two samples were carried out transcriptome sequencing. Selecting virulence-associated genes and prediction sRNA of S.agalactiae by transcriptome data analysis, verifying expression levels of virulence-associated genes that have been selected by fluorescent quantitative PCR technology; verifying the part sRNAs of prediction by RT-PCR method. The results are important clues for research virulence genes function and sRNA regulation mechanism of fish source S.agalactiae, the specific contents are as follows:1. S.agalactiae were cultured in 25℃and 37℃, respectively, the two samples transcriptome sequencing analysis showed that: the total number of expressed genes is1929 in 37℃ sample(Test), the total number of expressed genes is 1935 in 25℃ sample(CK). The total number of significantly different genes of gene expression is 673 between two samples, the number of differentially expressed genes of significantly upregulated is146, the number of differentially expressed genes of significantly down-regulated is 527.Pathway enrichment analysis showed that:1215 genes were annotated to 105 pathways of the KEGG, significant differences genes are 447; significantly enriched pathways are 40,the pathway of most significant difference include metabolism Pathways 、 biosynthetic pathways、repair Pathways and signaling pathways.2. Selecting out 7 virulence-associated genes from streptococcus agalactiae transcriptome, That are cylI、cylA、cylK、cylJ、cylG、sodA �'� PI-2b. First, cloning andbioinformatics analysis of 7 virulence-associated genes, then, verifying 7virulence-associated genes by fluorescent quantitative PCR. The results show that: 7virulence-associated genes can obtain full length by cloning; the expression trend of 7genes fluorescent quantitative PCR results and transcriptome sequencing results is same,only multiples of the relative expression levels is different. In general, the reliability of transcriptome results is higher.3. Predicting sRNA 167 from S.agalactiae transcriptome, verifying part of predicted sRNAs by RT-PCR. The results showed that: SAR-1、SAR-9、SAR-4、SAR-14、SAR-T fragment length is 178bp、139bp、110bp、165bp、111bp by cloning and cloned fragment can exact match to predicted sRNA sequence, respectively. Illustrating these sRNA exist in S.agalactiae ZQ0910.