Analysis Of Transcriptome And MiRNAs In Bothriochloa Ischaemum Under Drought Stress

Drought is one of the main environmental stress factors that limit plant growth,development and productivity.B.ischaemum is a perennial plant of the genus of Bothriochloa in Gramineae,originated in Asian and Southern Europe.And it grows widely in warm-temperate regions around the world.B.ischaemum is drought-tolerant and widely used for grazing,as it has high yield and is resistant to trampling by animals.Past studies on B.ischaemum have focused on productivity,nutritional value,physiological ecology,and genetic diversity,but little has been done on the molecular mechanism and the signaling pathways of B.ischaemum affected by drought stress.High-throughput Illumina sequencing technology was used for the first time to analyze the transcriptomes and micro RNAs(miRNAs)for leaves and roots of B.ischaemum under drought stress.By identifying commonly and differentially expressed genes(DEGs)and miRNAs in leaves and roots under drought stress,and on the basis,the MYB transcription factor was excavated and analyzed,we identified possible molecular mechanisms of B.ischaemum's response to drought stress,and these results may help to improve the germplasm of B.ischaemum to further increase drought resistance in this species.The main results are as follows:1.Through the analysis of transcriptional expression profile for leaves and roots of B.ischaemum seedlings under drought and normal growth conditions,we found there were 7989 differentially expressed genes in leaves and 15675 differentially expressed genes in roots(compared to the corresponding organs of the control groups),of these,4489 and 5010 genes were up-regulated genes in leaves and roots,respectively.There were 2012 differentially expressed genes(DEGs)that were shared between leaves and roots,1068 were up-regulated.We identified common and distinct biological processes and metabolic pathways involved in drought stress between the two tissues.Most notably,there was a dramatic up-regulation of genes involved in plant hormone signal transduction especially ABA signal transduction components and flavonoid biosynthesis enzymes or regulation factors in drought stress treated leaves.Therefore,these two cellular processes likely confer resistance to drought stress in B.ischaemum.Overall,our findings provided new insights into a mechanism involving the synergistic interaction between ABA signaling and secondary metabolism during the drought adaptation of B.ischaemum.2.Based on the transcriptome data of B.ischaemum,we used Illumina sequencing andbioinformatics methods to identify and analyze the expression of miRNAs in leaves and roots of B.ischaemum seedlings under drought stress and normal growth conditions.Seventy-nine Known miRNAs belonging to 20 families and 92 Novel miRNAs were identified from four libraries.There were 65 miRNAs(28 upregulated and 37 downregulated)and 27 miRNAs(15upregulated and down 12 downregulated)were differentially expressed in leaves and roots after drought stress,respectively.The major miRNAs responding to drought stress were miR156,miR164,miR166,miR169,miR172,miR396,miR398,miR408 and miR528.The enrichment analysis of GO biological function and KEGG metabolic pathway showed that plant hormone signal transduction was the main metabolic pathway in response to drought stress in leaves and roots,and flavonoid biosynthesis was the major rich factor for Bothriochloa ischaemum leaves responding to drought stress.3.Based on the obtained transcriptome data of B.ischaemum,the R2R3-MYB transcription factor family of B.ischaemum was excavated by comparing with MYB transcription factors of Arabidopsis thaliana genome.At the same time,their putative functions were predicted in combination with the phylogenetic tree and classified into subfamilies using At MYBs describing known functions.The results showed that 186 Bi MYB transcription factors were excavated,and they encoded 47-1062 amino acids.They were hydrophilic unstable proteins,with alpha-helix and irregular curl as the main secondary structural components.GO functional annotations indicated that they were mainly involved in biological processed such as growth and development,secondary metabolism and stress response.Sequence alignment of 43 R2R3-MYB transcription factors from B.ischaemum showed that the domain of their was highly conservative and contained the structure of[W]-X(19)-[W]-X(19)-[W]-X(n)-[W]-X(18)-[W].Finally,through the analysis of the heat map drawn by the data of the expression level of R2R3-MYB transcription factors in leaves and roots under drought and normal growth conditions,we screened out the R2R3-MYB transcription factors that may be involved in the response of B.ischaemum to drought stress,including Bi MYB129,Bi MYB64,Bi MYB67,Bi MYB108,Bi MYB142,Bi MYB143,Bi MYB35 and Bi MYB185.The identification of the functions of these genes will further enrich the drought-resistant gene resources.4.Based on an EST obtained by transcriptome sequencing of B.ischaemum,a c DNA sequence homologous to MYB transcription factor family of other plants was cloned by SMARTTM RACE,the homology of the gene with Sorghum bicolor reached 89%.It wasnamed Bi MYB which has 1343 bp and encoded 293 amino acids,It was predicated that the protein encoded by Bi MYB was located in the nucleus and was an unstable hydrophilic protein.We analyzed the expressions of the gene in different tissues and stress conditions by q RT-PCR,the results showed that the expression of the gene was different in different tissues,the highest in leaves and the lowest in roots.At the same time,the gene was induced by exogenous ABA and drought stress,which suggested that Bi MYB might participate in the response of B.ischaemum to drought stress through ABA signal transduction pathway.