Roles Of MiRNAs And Their Target Genes In Response And Adaptation Of Jatropha Curcas To Low Temperature

Jatropha curcas is a kind of energy plant species with great potential for development,and low temperature is a main limiting factor for production of J.curcas.The previous research from our laboratory showed that chill-hardening could enhance chilling resistance significantly.It is well-known that microRNAs play important roles in response and adaptation of plants to environmental stresses,but it is little known how miRNAs regulate chilling resistance in J.curcas.Understanding the molecular mechanism of miRNAs involved in the improvement of chilling resistance during chillhardening of J.curcas,and obtaining the key miRNAs and its target genes closely related to chilling resistance,are conducive to better elucidating molecular mechanisms of formation of chilling resistance in J.curcas.In this present study,screening and comparison of reference genes for miRNAs quantitative real-time PCR(qRT-PCR)in J.curcas under chilling stress was carried out firstly,then gene expression of some important and differentially expressed miRNAs genes and their corresponding target genes were analyzed through qPCR during the formation of chilling resistance induced by the chill-hardening,which will lay theoretical and experimental foundation for revealing the molecular mechanisms of the involvement of miRNAs in formation of chilling resistance in J.curcas during chill hardening.The results are as follows:1.Based on the previous results of small RNA-seq,11 candidate internal reference genes were selected from the cold-treated J.curcas and their expressions in different samples were detected by real-time fluorescence quantitative PCR technology.GeNorm,NormFinder and BestKeeper softwares were used for comprehensive analysis of expression stability.The results showed that the most stable expression genes were miR6448 and U6,and the Ct value of miR6448 was about 23,with moderate expression abundance.Ct value of U6 was about 10,indicating high expression abundance.Therefore,miR6448 can be used as a reference gene for the expression of miRNAs qRT-PCR with moderate abundance under low temperature stress of J.curcas.U6 can be used as an internal reference gene of miRNAs qRT-PCR with high abundance under low temperature stress of J.curcas.2.32 differentially expressed candidate miRNAs were detectedby qPCR under chill-hardening,and their expression trends were consistent with the small RNA-seq results except miR5284,miR5494 and miR6189.The results showed that the expression level of 22 miRNAs in J.curcas seedlings with chill-hardeGOing was higher than that without chill-hardening,and 8 miRNAs in J.curcas seedlings with chill-hardening demonstrated lower expression level during the following chilling stress treatment.There was no significant difference in the expression levels of the two miRNAs between the seedlings with and without chill-hardening.The results of qRT-PCR provided a certain experimental basis study of roles of some important miRNAs in the formation of chilling resistance induced by chill-hardening in J.curcas.3.Through bioinformatics analysis of differentially expressed 32 miRNAs,as well as predicted target genes,GO enrichment,Pathway analysis,and functional category were carried out.GO enrichment analysis of the 10 genes were involved in lipid metabolism process,84 genes involved in protein phosphorylation,signal transduction,and other biological processes;Pathway analysis received 212 results,12 in plant hormone signaling,12 in RNA transshipment,9 in fatty acid metabolism,and 5 in phospholipases.The predicted target genes were functionally classified into transcription factor,protein kinase,phosphatase,synthase,synthetase,peroxidase,catalase,reductase,hydrolase,phospholipase and desaturase.Fluorescence quantitative PCR was used to analyze the miRNA-mRNA expression patterns of 6 candidate miRNAs and their corresponding target genes during chill-hardening and following chilling stress in J.curcas seedlings.The results showed that miR5254 had a negative regulatory effect on the target gene PLC.miR5284 had a negative regulatory effect on the target gene FAD6.miR6189 had a positive regulatory effect on the target gene FAR.miR165 and miR4239 had negative regulatory effects on the target gene PLA.miR5827 had negative regulatory effects on the target genes FAD3.These results from miRNA-mRNA interaction indicated that miRNAs could be involved in regulation of their corresponding target genes to enhance chilling resistance in J.curcas.4.From 6 identified target genes which are closely related to membrane lipid component,miRNA-regulated and chilling-responsive,two genes(FAD3 and FAD6)with high expression level and significant variation were selected to carry out gene cloning,bioinformatics analysis and predict of protein structures.The results will be helpful for subsequent genetic engineering improvement of chilling resistance in J.curcas.