RNA-sequencing Analysis Of Sesame Transcriptome On Infection With The Ralstonia Solanacerun And Identification Of The AQP Gene Family In Sesame

Sesame(Sesamum indicum),with a high value of nutrition,is an important oil crop,but inhited both in yield and quality by bacterial wilt which is induced by Ralstonia solanacearum.Plant bacterial wilt disease has became the second largest plant pathogenic bacteria with a wide spread in temperate,tropic and sub tropic zones in the world.Bacterial wilt is hard to be prevented effectively because of its transfer through soil.It is meaningful to carry out researches about the interaction mechanis m between sesame and Ralstonia solanacearum,without any cultivar shows high-resistance to bacterial wilt in sesame.To identify the changes in global transcriptome of sesame after inoculation with the Ralstonia solanacearum,RNA was extracted from three different onset period sesame stem,combining two biological replicates each to increase reliability.Six cDNA libraries were constructed and sequenced separately using an Illumina HiSeq2500 genome analyzer.We obtained 13.5 Gb data of sequence,with a mean size of the 2.25 Gb per sample.The gene expression levels for the biological replicates seem to be mostly the same,implying that the Illumina sequencing solution was reproducible and reliable.After stringent quality assessment and data filtering,alignment was performed against the sesame reference genome available at the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences using the TopHat,and differential gene expression was analyzed using Cufflinks.A total of 4367 differentially expressed genes were identified,down-regulated gene always more than up-regulated gene at each stage.there are more differentially expressed genes in SO vs S1 than S1 vs S2,may be showed that early stage is critical for Ralstonia solanacearum to infect sesame.Through the cluster analysis of differentially expressed genes,all the genes were divided into 6 clusters,and the number of genes in different clusters was different.In based of cluster analysis,GO enrichment analysis is promoted by agriGO separately,and the function between different clusters have similarities an ddifferences,the clustering group with similar expression patterns is tend to be gat hered by similar function.The results of pathway enrichment analysis showed that the starch and sucrose metabolism,carbon metabolism pathways are involved in each group significantly while the plant hormone signal transduction,plant-pathogen inter action,cysteine and methionine metabolism,peroxisome and the amino acid biosynth esis pathways distributed in cluster2 and cluster5 meanwhile.Otherwise,The plant-pathogen interaction pathway also distributed in clusterl and cluster6.It had showed the avidence that the metabolism of amino sugars and nucleotides was inhibited by Ralstonia solaacearum for amino sugars and nucleotides metabolism pathway distributed in Cluserl and Cluser4.AQP gene sequences were isolated in sesame genome by bioinformatics combined with gene annotation information and verified by InterPro.A total of 36 AQP genes were identified in sesame genome.Sequence comparison and phylogenetic tree a nalysis suggested that these AQPs of sesame can be categorized into 5 subgroups:PIP(13 members),TIP(12 members),NIP(8 members),SIP(2 members)and XIP(only 1 member).34 AQP genes were mapped onto 12 linkage groups.The gene structure,protein sequence,subcellular location and conservation in amino acids in the same subgroup were similar.Under Ralstonia solanacearum challenge,the expression of some members in PIP and TIP were up or down regulated,but the expression of members in NIP,SIP and XIP had no significant changes.For instance,SiPIP1;2,SiPIP1;3,SiPIP2;3,SiPIP2;4 were up-regulated,among them,SiPIP1;3 and SiPIP2;3 retain constantly up-regulation,but SiPIP1;2 and SiPIP2;4 exhibit up-regulation at early infection stage and down-regulation at later stage.SiPIP1;4,SiPIP2;1,SiPIP2;6,SiTIP1;1,SiTIP1;3,SiTIP2;1 and SiTIP2;2 were drastically down-regulated.The e xpression of these genes were confirmed by qPCR,which showed similar results to that of transcriptome sequencing.The substrates transported by each group of AQP were predicted based on the types of ar/R selective filters and amino acid residues in Froger's position.Under Ralstonia solanacearum inoculation,expressions of some members in PIP and TIP were significantly up or down regulated.Gene expressi on under Ralstonia solanacearum inoculation show that some members in PIP and TIP may play crucial roles in sesame-Ralstonia solanacearum interaction.