Association Analysis Of Rice Cold Tolerance Genes And The Molecular Mechanism Of OsCTE3 Involved In Cold Stress

Rice is an important food crop,it is essential to improve its resistance to high and stable production.As a warm crop,low temperature stress is a major factor hindering its high and stable yield.Therefore,it is very important to cultivate rice resistant cultivars by using genome-wide association analysis to study cold tolerance genes,identifying cold-tolerant germplasm resources,and using transgenic methods to identify cold tolerance genes and study the molecular mechanisms involved in cold stress.This paper is divided into two parts to analyze the cold tolerance varieties,the correlation analysis and mining cold tolerance genes,study the molecular mechanism of cold tolerance gene by molecular biology method,the main results are as follows:1.In this study,582 rice resources were used for genome-wide association analysis,to identify cold-tolerant and cold-sensitive varieties,and excavate the candidate cold-resistant genes:1.1 According to the phenotypic data,572 germplasm resources were divided into three grades.There were 23,64 and 61 species belonged to highly resistant,cold and cold sensitive germplasm resources.1.2 Two cold tolerance genes were identified in the japonica rice germplasm community,which were the cytochrome P450-related gene LOC_Os01g50490 and the stress-resistant gene LOC_Os06g36240.1.3 A total of 59 cold tolerance candidate genes were identified in the indica rice population,and 36 of them were located on chromosome 11,17 of which were related to rice disease resistance.Among them,four cloned candidate genes related to the abiotic stress of rice were very important,such as the MYB family gene LOC_Os 11 g47460 involved in endogenous new hormones and abiotic stress;cold-related genes LOC_Osllg47500 and LOC_Os11g47820;overexpressing plants for biological and Abiotic stress showed resistance gene LOC_Os03g29760.Another two cold-resistant candidate genes,LOC_Os11g47760 and LOC_Os09g20090,were associated with other stress.1.4.149 candidate genes were mapped in the in the whole population of germplasm resources.A very important clonal cold tolerance gene LOC_Os12g40550 was located,which involved in ABA dependent signal transduction of cold tolerance and was a critical low temperature response gene that regulated the degradation of ureide in low temperature stress.To prove that the study is effective and reliable.Another 3 important cold tolerance candidate genes was located,LOC_Os08g4053 promoter with cis-element related to drought resistance,cold tolerance and ABA signal transduction;LOC_Os04g37619,involved in ABA synthesis pathway,induced by low temperature of anthers,and differentially expressed in cold sensitive and cold tolerant rice;The temperature related gene LOC_Os12g40540 was up-regulated at room temperature and decreased in high temperature.Another 5 candidate gene LOC_Os02g08230,LOC_Os02g12580,LOC_Os02g12800,LOC_Os04g37630 and LOC_Os11g34440 were associated with other stress resistance.2.In this paper,we analyzed the molecular mechanism of cold related gene OsCTE3 in cold stress by phenotypic identification,hormone treatment,yeast two-hybrid and in vitro ubiquitination experiments:2.1.Phenotypic identification showed that OsCTE3 could be induced by cold stress,and the expression level was significantly up-regulated within 48 hours after cold treatment.Over-expression OsCTE3 showed cold sensitive and RNAi was cold tolerant.And cold tolerance or cold sensitive phenotype was related to the expression of OsCTE3 in transgenic plants,the more expression of OsCTE3,the more cold sensitive,the less the more resistant to cold.2.2.Hormone identification experiments showed that Over-expression OsCTE3 was insensitive to ABA.After being treated with exogenous ABA,the stem length and root length of interfering plants were significantly different from those of wild type.Fluorescence quantitative results showed that the expression of OsCTE3 also changed with different concentrations of exogenous ABA.2.3.OsCTE3 can be induced by ABA and cold stress at the same time,suggesting that OsCTE3 may be involved in the cold stress response of ABA-dependent signaling pathway.2.4.Six candidate genes,OsRNS4,OsDi19-5,Os03g0681400,OsRBCs5,Os03g0784800 and Os05g0187000,were screened by yeast two-hybrid experiments.The results showed that they interacted with OsCTE3 in the yeast strain.OsCTE3 may be involved in the cold stress response by synthesizing and degrading OsRBCs and OsRNS4 protein through ABA signaling and 26S proteasome pathways.2.5.In vitro ubiquitination experiments showed that OsRBCs is a ubiquitination activity of E3 ubiquitin ligase.