水稻OsCLF基因的功能分析及其对铝毒胁迫的响应  

Functional Analysis of Gene OsCLF and Its Response to Aluminum Stress in Rice(Oryza sativa)

作　　者：李超 肖恩宗 白庆 徐枫 冀曦悦 李春娥 王玉琪 LI Chao;XIAO En-Zong;BAI Qing;XU Feng;JI Xi-Yue;LI Chun-E;WANG Yu-Qi(School of Environmental Science and Engineering,Guangzhou University,Guangzhou 510006,China;Shanghai Fuxing Senior High School,Shanghai 200434,China)

机构地区：[1]广州大学环境科学与工程学院,广州510006 [2]上海市复兴高级中学,上海200434

出　　处：《农业生物技术学报》2022年第5期837-846,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(41877423);广东省科技厅乡村振兴战略(620096-3);广州市科技计划(202102010385)。

摘　　要：组蛋白甲基化修饰在真核生物基因表达调控中发挥着重要的作用。调控拟南芥(Arabidopsis thaliana)卷叶的蛋白(curly leaf protein, CLF)是重要的组蛋白甲基转移酶,参与多种细胞发育过程。本研究对水稻(Oryza sativa) OsCLF基因的功能进行了初步研究。通过双酶切克隆,构建了OsCLF基因过量表达载体pCAMBIA1300-35S-OsCLF,进而转化拟南芥clf突变体。表型及生化功能分析表明水稻OsCLF蛋白具有类似拟南芥CLF蛋白的H3K27三甲基转移酶的功能,且能功能互补使拟南芥clf突变体的表型为野生型。qPCR检测发现,水稻OsCLF基因的表达在长日照条件下较高,且铝毒胁迫能显著诱导其表达;随后将RNA干涉载体pYLRNA-OsCLF-RNAi转入水稻,长日照条件下的表型分析表明,OsCLF基因RNAi水稻株系的株高、旗叶长宽度、花粉活力及结实率均显著低于野生型水稻(P<0.01),而分蘖数和叶绿素含量则显著高于野生型水稻(P<0.01);下调OsCLF基因表达的RNAi水稻株系对铝毒胁迫的抗性得到提高,说明OsCLF负调控水稻耐铝基因的表达。本研究初步揭示了OsCLF基因参与的水稻生长发育的生物学过程,为进一步探明这些过程的表观遗传机理提供参考依据。Histone methylation plays an important role in regulating gene expression in eukaryotes.The curly leaf protein(CLF),which regulates leaf curling in Arabidopsis,is an important histone methyltransferase and involved in a variety of cell development processes.In this study,the functions of OsCLF were preliminarily studied in rice(Oryza sativa).The overexpression vector pCAMBIA1300-35S-OsCLF was constructed and identified by double restriction endonuclease digestion and sequencing,and then transformed into Arabidopsis thaliana clf mutant.Phenotypic and biochemistry function analysis revealed that OsCLF of rice had the histone H3K27 tri-methyltransferase function,which was similar to CLF of A.thaliana,and the phenotypes of clf mutant could be functional complemented to wild type's by OsCLF.Fluorescence quantitative PCR analysis results showed that the relative expression level of OsCLF was higher under long-day condition than that in short-day condition,and its expression was significantly induced by aluminum stress(P<0.01).After that,the RNA interference vector pYLRNA-OsCLF-RNAi was transferred into wild type rice.Phenotype analysis indicated that the plant height,flag leaf length and width,pollen viability and seed setting rate of OsCLF-RNAi transgenic plants were all significantly decreased,but the tiller number and chlorophyll content were remarkablely increased than those of wild type rice(P<0.01).In addition,the OsCLF RNAi transgenic rice lines were more tolerant to aluminum stress than wild type rice,which indicated that OsCLF negatively regulated aluminum tolerance related genes'expression.This study preliminarily revealed the biological processes of rice growth and development involved by OsCLF gene,and provides a reference basis for further study on the epigenetic mechanism of these processes.

关 键 词：水稻 OsCLF基因 过表达 RNA干扰 

分 类 号：S511[农业科学—作物学] S188