Identification And Functional Analysis Of MiRNA Responsive Stress Of High Temperature In Rice(Oryza Sativa L.)

With the aggravation of global climate,People are becoming more and more attention to the harm of high temperature on crop.In recent years,the high temperature harm to rice production in the Yangtze basin has become the one of the major problems.But the studies level of high temperature stress to rice in the molecular is lack.Relative to other abiotic stress in rice is still in its infancy.More and more evidences have indicated miRNA as the key regulation factor in the process of plant abiotic stress response plays an important role in rice growth and response to stress.The research about miRNA has become a hotspot in the field of rice molecular genetic manipulation.The understanding of the mechanism of miRNA about rice abiotic stress response is constantly deepening.At present,the most of studies about rice stress resistance focus on low temperature,drought,high salt and heavy metal stress.A few studies focus on high temperature stress.Therefore,the research about miRNA of response to high temperature stress in rice aimed to provide a new theoretical support for the selection of high temperature resistant varieties and the discovery of anti stress genes through making clear the mediated pathway in response to high temperature stress of miRNA and its target genes.The following is the main results of this paper.1)Two small RNA libraries were constructed from normal control group(NT)and high temperature stress group(HT)of Nagina22-19379(N22).The libraries were submitted to high-throughput sequencing.Received a total of nearly 5 million sRNA fragments.The length of fragment is mainly distributed in the range of 20-24 nt,of which 24 nt sRNA is the main type,followed by sRNA sequence of 21 nt and 23 nt.The other length of fragment is relatively low.The results are consistent with previous studies.685 conserved miRNAs,539 new miRNAs,and 258 differentially expressed miRNAs were identified.Of which 122 differentially expressed miRNAs reached a very significant level.At same time,the results of qPCR analysis in ten miRNAs were consistent with sequencing.2)A degradome deep sequencing library was constructed from N22.Thelibraries were submitted to deep sequencing by using the Illumina Solexa system.Then the identified 129 differentially expressed miRNAs in the high-throughput sequencing and degraded mRNA sequences in degradome library were used for data analysis.190 targets of 46 miRNAs were identified.They were involved in plant growth and development,response to stress,metabolism,signal transduction and other plant physiological processes.Most of them are transcription factors,and the other are metabolism related genes,transporter protein,DNA/RNA binding protein,reverse transcriptase protein,zinc finger protein,cell membrane binding transfer protein and some unknown proteins.these proteins play an important role on normal growth and development of rice.GO function enrichment analysis of identified target genes of high temperature miRNA indicated that they were enriched in DNA-dependent transcription,auxin mediated signaling pathway,flower and seed development,CCAAT-binding factor complex,enzyme binding,metal ions binding and metal ion tras-membrane transport activity.KEGG pathway analysis showed 53 target genes of high temperature miRNA were involved 28 pathways,and were significantly enriched the pathways of antigen processing pathway,antigen processing and presentation,MAPK signaling pathway,terpenoid backbone biosynthesis and glutathione metabolism.qPCR analysis of some target genes showed the expression pattern of target gene was basically the same as that of miRNA.3)The over-expressing vector of miR162 a was constructed.Using the genetic transformation system of rice,we successfully obtained 15 transgenic plants of over-expressing miR162 a.qPCR analysis showed that the expression of miR162 a in transgenic plants was substantially increased,and the expression of OsDCL1 was significantly decreased.Phenotypic observation of transgenic T0 generation plants showed that the growth was not obvious difference in the seedling stage between transgenic rice and wild type rice.However,compared with the transgenic rice,the growth of wild rice was faster and more vigorous.But in the reproductive growth stage,there were bleaching of the grains in wild type rice,and the filling and plumpness of grains decreased significantly comparing with the transgenic rice.That may be due to a decrease in OsDCL1 expression,which activated the immune response of plant cells.The results of determination physiological indexes further showed the over-expressing miR162 a increased high temperature in a certain extent.In this study,miRNA of high temperature response were obtained by high-throughput sequencing.Their target genes were verified by degradation sequencing,and the functional annotation and enrichment of these target genes were analyzed.Over-expressing miR162 a increased high temperature stress tolerance in a certain extent.The results indicated that miRNA played an important role in high temperature tolerance in rice.