茶树Ankyrin基因启动子的克隆及其5′UTR内含子功能  

Isolation of the Ankyrin gene promoter from tea plant(Camellia sinensis L.) and a subsequent analysis of the function of its 5′UTR intron

作　　者：郑世仲 江胜滔 陈美霞 林玉玲[3] 赖钟雄[3] 林金科[4] ZHENG Shizhong;JIANG Shengtao;CHEN Meixia;LIN Yuling;LAI Zhongxiong;LIN Jinke(College of Life Sciences,Ningde Normal University,Ningde 352100,China;Institute of Fujian Higher Education for Local Bio-resources in Ningde City,Ningde 352100,China;Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Anxi College of Tea Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]宁德师范学院生命科学学院,宁德352100 [2]闽东特色生物资源福建省高校工程研究中心,宁德352100 [3]福建农林大学园艺植物生物工程研究所,福州350002 [4]福建农林大学安溪茶学院,福州350002

出　　处：《应用与环境生物学报》2019年第6期1381-1387,共7页Chinese Journal of Applied and Environmental Biology

基　　金：福建省高校杰出青年科研人才培育计划项目资助~~

摘　　要：为了解启动子在茶树Ankyrin基因(CsAnkyrin)表达调控中的作用,以茶树新品系‘1005’嫩芽为材料,通过染色体步移技术克隆CsAnkyrin基因启动子序列,利用Neural Network Promoter Prediction和PlantCARE在线软件等对启动子序列进行生物信息学分析,将含有内含子和删除内含子的启动子序列(+intron/-intron)分别定向替换pCAMBIA1301表达载体的CaMV35S启动子,构建重组表达载体后分别转化拟南芥,并进行GUS组织化学染色分析.结果显示,克隆得到的CsAnkyrin启动子序列(命名为proAnk)为1010 bp,含有一个5′UTR(5′Untranslated regions,5′非编码区)内含子.启动子序列除含有启动子核心元件TATA-box和CAAT-box,还存在有激素响应元件、光响应元件、厌氧胁迫应答元件以及大量功能未知或功能特异的顺式作用元件.删除5′UTR内含子后的启动子序列能够驱动下游GUS报告基因表达,保留5′UTR内含子的启动子序列不能驱动下游GUS报告基因正常表达,但是RT-PCR实验结果表明5′UTR内含子在转录后没有被切除,而是和GUS基因一起转录产生融合mRNA.本研究表明,proAnk具有诱导型启动子特性,5′UTR内含子对下游基因正常表达具有负调控作用,且可能是在翻译水平上对下游基因表达产生影响.(图7表1参32)In this study,we aimed to elucidate how the process of CsAnkyrin expression is regulated in tea plant.Briefly,a 1010 bp 5′-flanking sequence of CsAnkyrin(proAnk)was isolated from the shoots of the tea cultivar,1005,using the genome-walking method.Thereafter,sequence analysis was carried out using online bioinformatics resources,such as Neural Network Promotor Prediction and PlantCARE,to reveal its basic core promoter,transcription start site,and cis-regulatory elements.We found that some of the typical cis-acting elements,such as the TATA-box and CAAT-box elements,some of the cis-elements that respond to hormone,light,and stress,and other cis-elements with unknown or a specific function were contained within proAnk,suggesting that it is an inducible promoter.Additionally,we identified a 5′UTR intron within the proAnk sequence.The CsAnkyrin promoter sequences with or without an intron sequence(+intron/-intron)were cloned into the expression vector,pCAMBIA1301,to replace the CaMV35S promoter and produce a new recombinant expression vector,respectively.The recombinant plasmid was then transformed into Arabidopsis thaliana using the floral dip method.Based on GUS histochemical analysis of transgenic A.thaliana,the proAnk including the 5′UTR intron could not drive the expression of the GUS gene,while those excluding the 5′UTR intron could drive its expression.Semi-quantitative RT-PCR also revealed that the 5′UTR intron was not removed during the transcriptional process;instead,it was transcribed to mRNA.Such finding suggests that the 5′UTR intron of the CsAnkyrin gene negatively regulates downstream gene expression and may function in the process of translation.

关 键 词：茶树 CsAnkyrin 启动子 顺式作用元件 5′UTR内含子 

分 类 号：S571.1[农业科学—茶叶生产加工] Q78[农业科学—作物学]