橡胶草乳管特异性高表达基因TkREF的启动子区段鉴定  

The Promoter Regions Identification that Regulates the TkREF Specific and High Expression in Latex

作　　者：刘星[1,2] 杨雪 张芳[1,2] 唐朝荣 刘开业 Liu Xing;Yang Xue;Zhang Fang;Tang Chaorong;Liu Kaiye(Natural Rubber Provincial and Ministerial Collaborative Innovation Center,College of Tropical Crops,Hainan University,Haikou,570228;Hainan Yazhou Bay Seed Laboratory,Sanya Nanfan Research Institute of Hainan University,Sanya,572025)

机构地区：[1]海南大学热带作物学院,天然橡胶省部共建协同创新中心,海口570228 [2]海南大学三亚南繁研究所,海南崖州湾种子实验室,三亚572025

出　　处：《分子植物育种》2025年第4期1154-1163,共10页Molecular Plant Breeding

基　　金：国家自然科学基金项目(No.32201450,No.31825007);海南大学高层次人才启动费项目共同资助。

摘　　要：橡胶草(Taraxacum kok-saghyz Rodin,TKS)根部天然橡胶含量可达干重的20%以上,且橡胶的质量和特性与橡胶树相似,因此被认为是理想的产胶替代植物。橡胶延伸因子(rubber elongation factor,REF)是胶乳中特异高表达且对橡胶生物合成具有重要作用的一类蛋白,然而调控其乳管特异性高表达的启动子区段尚未被鉴定。本研究利用实时荧光定量PCR(RT-qPCR)分析了TkREF的时空表达特性,结果发现该基因在橡胶草生长的不同阶段均表现为胶乳特异性高表达,且表达水平呈现逐渐增加的趋势。启动子顺式作用元件预测分析发现,TkREF的启动子区主要包含环境响应元件(17%)、功能未知元件(44%)和启动子基本元件(36%)。对TkREF不同长度启动子区段驱动GUS报告基因的转基因橡胶草进行GUS染色和GUS酶活性分析,结果表明在TkREF启动子-1088 bp至-759 bp之间存在调控该基因在胶乳中高表达的顺式作用元件,而在-1980 bp和-1088 bp之间以及-759 bp和-359 bp之间可能存在抑制该基因胶乳表达的顺式作用元件。本研究为阐明TkREF参与产胶调控的机制及其在橡胶草高产分子育种中的应用提供了理论依据。The rubber dandelion(Taraxacum kok-saghyz Rodin,TKS)is an ideal alternative crop for natural rubber production since its rubber content of root can be more than 20%of its dry weight and its rubber quality and characteristics are similar to rubber tree.Rubber Elongation Factor(REF)is a kind of protein that is highly expressed in the laticifer cells and plays an important role in the biosynthesis of natural rubber.However,the promoter region that regulates its high and specific expression in laticifer cells has not been identified.In this study,real-time fluorescence quantitative PCR(RT-qPCR)was used to identify the spatio-temporal expression pattern of TkREF,and proved that its highly and specifically expressed in laticifer cells at different growth stages.Moreover,the expression level of TkREF showed a gradually increasing trend in the whole growth period.The prediction analysis of cis-acting elements promoter showed that the promoter region of TkREF,containing cis-acting elements with environmental response(17%),unknown function(44%),and promoter basic elements(36%).GUS staining and GUS enzymatic activity analysis of transgenic rubber dandelions driven by different length promoter seg ments of TkREF showed that the region of-1088 bp to-759 bp contains cis-acting elements that regulate its high expression in latex,while the regions of-1980 bp to-1088 bp and-759 bp to-359 bp contain cis-acting ele ments that down-regulate its expression in latex.This study provided a theoretical basis for elucidating the mechanism of TkREF involved in the regulation of rubber production and its application in high-yielding molecular breeding of rubber dandelion.

关 键 词：TkREF 乳管特异性 启动子区段鉴定 GUS酶活 GUS染色 

分 类 号：S794.9[农业科学—林木遗传育种]