东乡野生稻苗期耐冷QTL qCTS3.3候选基因LOC_Os03g53670-DX在低温胁迫下的表达及克隆  

Expression Analysis and Cloning of Cold-tolerant QTL qCTS3.3 Candidate Gene LOC_Os03g53670-DX in Dongxiang Wild Rice at Seedling Stage under Low Temperature Stress

作　　者：沈春修[1] 陈丽 却志群[1] 卢其能[1] SHEN Chunxiu;CHEN Li;QUE Zhiqun;LU Qineng(College of Life Sciences,Resources and Environment Sciences,Key Laboratory of Regulation of Crop Growth and Development in Jiangxi Province,Yichun University,Yichun 336000,China)

机构地区：[1]宜春学院生命科学与资源环境学院,江西省作物生长发育调控重点实验室,江西宜春336000

出　　处：《云南农业大学学报（自然科学版）》2020年第4期562-571,共10页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(31660379);江西省教育厅科学技术研究项目(GJJ180845);江西省作物生长发育调控重点实验室开放课题项目(KFJJ201804)。

摘　　要：【目的】研究东乡野生稻苗期耐冷QTL qCTS3.3候选基因的功能。【方法】结合课题组前期完成的转录组分析数据,将落在qCTS3.3 QTL区域的东乡野生稻苗期耐冷相关差异表达基因LOCOs03g53670-DX作为qCTS3.3的候选基因,通过实时荧光定量PCR验证不同冷胁迫处理下东乡野生稻中该基因表达情况,并利用RT-PCR技术从东乡野生稻幼苗中扩增LOCOs03g53670-DX的全长cDNA,并进行该基因的过表达载体构建。【结果】测序结果表明:LOCOs03g53670-DX基因的cDNA全长为1 986 bp,共编码661个氨基酸,蛋白分子量为173 ku,理论等电点(pI)为4.92。在线分析软件预测LOCOs03g53670-DX氨基酸序列近C端包含1个高度保守结构域YTH,表明该基因属于RNA结合蛋白。系统进化分析表明:LOCOs03g53670-DX与高粱(XM021447002.1)和菠萝(XM020225621.1)的YTH结构域基因位于同一进化分支上,亲缘关系最近。进一步利用农杆菌介导转化方法,经潮霉素筛选及转基因苗叶片GUS化学染色阳性鉴定,最终得到了转入LOCOs03g53670-DX基因的26株阳性转基因植株。【结论】本研究为后续LOCOs03g53670-DX基因在冷胁迫条件下的功能和作用机制研究奠定材料基础。[Purpose]In order to study the functional mechanism of cold-tolerant QTL qCTS3.3 candidate gene LOCOs03g53670-DX in Dongxiang wild rice at seedling stage under cold stress.[Method]The differentially expressed gene LOCOs03g53670-DX located in qCTS3.3 QTL region was used as candidate gene for qCTS3.3 based on the analysis of transcription data. Fluorescence quantitative PCR was used to verify the expression of this gene in Dongxiang wild rice with different time lengths of cold stress based on the analysis of transcription data. The full length cDNA of LOCOs03g53670-DX gene was cloned from Dongxiang wild rice by RT-PCR with the designed primers according to the corresponding locus sequence information of Nipponbare in the public database. The over-expression vector was then constructed successfully.[ Result]Sequence analysis showed that the full length cDNA of LOCOs03g53670-DX was 1 986 bp and encoded a protein about 173 ku which was composed of 661 amino acids. Its corresponding theoretical isoelectric point(pI) was 4.92. It was predicted that the C-terminal amino acid sequence of LOCOs03g53670-DX contained one highly conserved structure domain YTH using the online analysis software. It was suggested that the gene was a member of RNA binding protein gene family. Phylogenetic analysis results showed that the LOCOs03g53670-DX had the closest relationship with Sorghum bicolor(XM021447002.1) and Ananas comosus(XM020225621.1). Then the over-expression vector of the gene LOCOs03g53670-DX was transferred by Agrobacterium-mediated method, and 26 LOCOs03g53670-DX transgenic rice plants were obtained after hygromycin selection and GUS histochemical staining detection.[Conclusion]This study laid an important material base for further study on the functional mechanism of gene LOCOs03g53670-DX under cold stress.

关 键 词：东乡野生稻 苗期耐冷性 荧光定量PCR YTH域 生物信息学 

分 类 号：S511.034[农业科学—作物学]