| Jatropha curcas,a member in Euphorbiaceae,is one of the important bioenergy plants,with the quality agro characters,such as resistance to drought and arid,and multiple usage like a fungicide and an anti-cancer drug.Due to the global climate changing,drought is more and more serious to agriculture.Therefore,responding to the climate change and to the food security strategy,it is effective to understand the mechanism of the drought resistance of J.curcas and the candidate genes.Our lab found that J.curcas seedlings increased the contents of proline,glycinebataine and soluble sugars to decrease osmotic potential and water potential after air dought,and all materials mentioned formerly were higher in leaves than those in stems.Later,the research of our lab indicated that J.curcas seedlings increased the solutes mentioned above to improve their drought resistance after drought hardening.For proline,its mechanism was that the activities of the key enzymes of the biosynthesis pathway were improved and depressed the enzymes activities of its catabolic pathway in J.curcas seedlings.Based on the former results,this research was to investigate the mechanisms of the regulation of miRNA during the rapid acclimation of drought after hardening.The main subject of this thesis is to study the mechanism of a rapid acclimation to drought stress after a period drought hardening in J.curcas.Based on the previous research,the experiment conditions were 15% PEG6000 for 48 h as drought hardening and followed air drought for 72 h as drought stress under 25 ?,16 h light and 75% relative humidity in artificial climate chambers.The time points for samples were during drought hardening 0 h(CK),6 h,12 h,24 h and 48 h and during drought stress 24 h,48 h and 72 h.In addition,the plants growing in the climate chamber without PEG600 treatment for 48 h and then with air drought for 72h(CK72),simultaneously.All the treatments had 3 replications and 27 samples as a total.RNAs extracted from the samples were sequenced for miRNAomes and transcriptomes with Illumina HiSeq2000 platform.The correlation analysis between samples showed that the transcriptomes of drought hardening samples in 12 h was not different from that of CK00,and the plant genes transcriptome changed greatly after hardening 24 h.After 48 h of hardening,the samples were more consistent with the drought control and the drought stressed samples,indicating that the plant had adjusted the genes transcriptions to adapt to drought stress.The transcriptome between CK72 and CK00 was very different,the drought-stressed samples were more consistent with CK72,and the hardening samples were more consistent with CK00.Therefore,it was more appropriate to CK00 as the control for drought hardening samples and CK72 as a drought stress control.After the raw reads of the high throughput sequencing were filtered to remove the dirty reads,more than 317 million clean reads were obtained for the microRNAomes,and 322270640 clean reads obtained for the transcriptomes.The sequences of microRNAomes were aligned with those of Jatropha genome,miRBase database and Rfam database.To predict the secondary structure of miRNA,the software of miReap,RNA Structure and other tools were used in combining the remained candidate miRNA sequences with the genomic sequence.As a result,2907 known-miRNAs and 2673 novel-miRNA were obtained.After differential expression analysis,the differentially expressed known-miRNAs could be merged into 52 known-miRNAs belonging to 22 gene families and 15 novel-miRNA based on the sequence of mature miRNAs.After normalization of the transcriptome gene expression data with FPKM,the expressions of genes in the air drought samples were compared with those of CK72,and the expressions of genes in the drought hardening samples were compared with those of CK00 to calculate the change fold.Took the logarithm of the change fold and record it as FoldChange.After removing the gene with a small absolute value of FoldChange with a threshold of 1.5,a total of 485 non-redundant differentially expressed genes were obtained.In addition,CK72 and CK00 were compared,and 667 genes were differentially expressed between them.The expression changes of these genes were caused by the difference of the growth of the two groups for a total of 5 days and the air drought after 3 days.The 485 non-redundant differentially expressed genes obtained from drought exercise and air drought treatment were compared with the differentially expressed genes of the two groups of CK;after removing the differentially expressed genes in the two controls,285 differentially expressed genes were remained;and then removing genes with small FPKM,106 differentially expressed genes were got.Based on bioinformatics analysis to the target genes of differentially expressed miRNA,and combined all the annotations with GeneBank,Gene Ontology and KEGG,it was found that the differentially expressed miRNA regulated the status of plants in 4 aspects: a)Regulation of genes transcriptions and translation.Firstly,miRNA regulating transcription factors could replace or compensate hormones in some extent to regulate genes expression.Secondly,miRNA regulated genes transcriptions of key enzymes to adjust some other targets genes of these enzymes.Thirdly,miRNA regulated some key enzymes and/or targets of organelle.b)Transduction of drought signals with miRNA-phosphoinositol pathway and Ser/Thr protein kinase.c)Modification of components of cytoplasm with the regulation of lipid metabolism.d)ROS regulation.All mentioned above could make a complicated regulation network for drought response commonly and interactively.After the data were normalized by TPM(>15)of the miRNA and FPKM(>15)of the target gene,the FoldChange of the miRNA and the predicted target genes were calculated.The miRNA,with FoldChange to be opposite to the predicted target genes FoldChanges,were selected.The target genes of differentially expressed miRNAs in this study were initially identified,and 12 pairs of miRNA-target genes were found;but the further verification were needed.Subsequently,the differentially expressed novel-miRNA data and target gene prediction results were combined,and then pre-jcu-miR56 was selected.The primers for cloning pre-jcu-miR56 were designed based on the vector pYES2.The combination vectors were introduced into Saccharomyces cerevisiae “INV SC1”.The wild-type S.cerevisiae INV SC1 was tested to determine the treatments strengthen of drought,high temperature and high salt stress.The stress conditions were determined to be 45 °C as high temperature stress and 8% NaCl(1.37 mol/L)as high salt stress.The four recombinant strains verified by sequencing and the two controls of pYES2 and wild type INV SC1 were subjected to the above-mentioned stress treatments,and it was found that under high temperature and high salt conditions,the growth of the strain introduced into pre-jcu-miR56 was better than the other strains.In order to analyze the reason for these differences,this study used yeast identification microplates to observe the changes in carbon source utilization of recombinant yeasts.The yeasts with pre-jcu-miR56 increased 18 available carbon sources.The increased carbon sources included six sugars and their derivatives,six organic acids and their derivatives and six amino acids and their derivatives.The organic acids and their derivatives might be related to enhancing lipid metabolism.In this study,a rapid research method was invented to investigate miRNA function.That was the combination of traditional gene transduction with the identification of carbon source.This approach shortens the initial functional study of miRNA to 7-14 days.With this method,the utilization of carbon sources was observed.This study preliminarily speculated that in yeasts,jcu-miR56 might be involved in the regulation of lipid metabolism.With transduction the precursor of jcu-miR56 into Arabidopsis thaliana and using genetic modified A.thaliana as controls,it was found that jcu-miR56 could improve the drought resistance of A.thaliana and could promote A.thaliana seeds of to germinate fastly under nomal and drought conditions.In J.curcas,catalase 2 was predicted as one of the targets of jcu-miR56.So,in A.thaliana,genes encoding catalases might be the targets of this miRNA. |
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