RNA-seq-Based Transcriptomic Analysis,cloning & Functional Characterization Of Key Genes Involving Drought Stress Adaptation And Responses In Jatropha Curcas

Jatropha curcas is an energy plant with great potential for exploration.Drought is an important environmental factor restricting the development and cultivation of J.curcas.Therefore,it is becoming an urgent task to elucidate the molecular mechanism underlying drought responses in J.curcas,which will provide detailed information on genetic breeding strategies aimed at improving traits with adaptation and tolerance to drought stress.Here,the drought responsive process in J.curcas leaves was investigated over a time course including drought hardening and air drought stress,using Illumina RNA-Seq.Then based on the real time PCR verification,two candidates with differential expression under drought stress were isolated,JcLEA and JcGLIP,with further functional characterization in Saccharomyces cerevisiae.These data provide a comprehensive dataset available for drought adaptation and responses in Jatropha,and will serve as a useful resource for genetic improvement of J.curcas.The main results are shown as follows:1.Three-week old J.curcas seedlings were used as materials,exposed to 10% PEG for drought hardening?0 h,6 h,12 h,24 h,48 h?,followed by air drought stress?24 h,48 h,72 h?.The seedlings without hardening-treatement were also transferred to 3-day air drought exposure,designated as control.Then the leaves were collected for RNA-seq analysis using Illumina Hiseq2000 platform.Three biological replicates for each sample?harvested from 5 individual plantlets?were sequenced independently.After subtracting the low-quality reads,each cDNA library produced an average number of clean reads at 11,933,126.A total of 26,979 candidates with annotation were obtained,by alignment to the reference genome and multiple databases.2.The NOISeq method was used to detect differentially expressed genes?DEGs?between selected comparisons.When exposed to drought hardening for 6 h,12 h,24 h and 48 h,compared with CK?0 h?,a total 27?67?74?186 DEGs were repressed,while 36?76?122?166 DEGs were induced.During the air drought stress treatment?for 24 h,48 h,72 h?,the number of down-regulated genes were 671?742?696 respectively,and 411,409,375 DEGs were up-regulated.A total of 1,573 drought responsive genes were identified,of which,most of the up-regulated genes related to osmotic substances and proteins production?the biosynthesis and signaling of phytohormone?antioxidant system and transcription factors.3.To reveal the major functional categories represented in the DEGs,the total 1,573 DEGs were subjected to KEGG and gene ontology enrichment analysis.The results showed that drought hardening promoted transcriptional regulation and hormone signal regulation,as a large number of transcription factors?such as WRKY,bHLH,NF,C3 H,HD-ZIP,etc.?and components related to hormone biosynthesis and signal transduction?such as SAMS,ACO,ARPs,GA20 oxidase,SnRK,ZEP,etc.?were up-regulated.In addition,drought hardening-induced genes were also widely involved in the biosynthesis of osmotic substances?such as galactose,trehalose and betaine?and stress-responsive proteins?such as ns LTPs,HSPs?,antioxidant systems including GST,GPX,?CER,WSD,GPAT,FAD,cytochrome P450,etc.?and biochemical pathways associated with carbohydrate metabolism and conversion?such as RuBisco,PGK,etc.?.4.To verify the reliability of the gene expression profiles obtained from RNA-seq data,qRT-PCR was performed on randomly selected 22 genes.The mean pearson correlation coefficient is 0.8 or more,indicating that RNA-seq data is a reliable reliable reflection of transcript abundance.5.LEA protein is a kind of protein closely related to stress resistance,present in many species oof higher plants.Our RNA-seq analysis indicated that the expression of six LEA-encoding transcripts was induced by drought treatment,of which,one candidate?XM₀12232372.1?showed a relatively highest basal expression level and a remarkable induction when exposed to drought stress.Sequence analysis showed that the CDS sequence of the gene was 459 bp and encoded a protein with 152 amino acids.When subjected to eolutionary analyses with the Nighbor-Joining method,it was classified to the fourth group of typical hydrophilic LEA protein?designated as JcLEA4?.Real-time quantitative PCR analysis showed that the gene was significantly affected by drought stress and ABA induction in the leaves of J.curcas,and its heterologous expression enhanced the tolerance of Saccharomyces cerevisiae to PEG treatment.6.GDSL esterase / lipase represents a newly discovered subfamily member of lipase with GDSL conserved domain.On the basis of RNA-seq analysis,we isolated a gene encoding GDSL hydrolase / esterase?named JcGLIP?from J.curcas.Following a conserved domain database?CDD?BLAST search,we identified that it was a member of SGNH hydrolase superfamily / subfamily according to conservative domain analysis.The CDS sequence of JcGLIP was 459 bp and encoded by 367 amino acids.The results of fluorescence quantitative PCR showed that they were induced by drought stress and ABA treatment.And it was a drought-fast response gene.Transgenic yeast also showed an increased resistance to PEG.All these findings suggest that JcLEA4 and JcGLIP may positively regulate ABA signaling and drought stress tolerance in J.curcas.