铁皮石斛DoAGL6基因及启动子克隆与分析  

Cloning and Analysis of DoAGL6 Gene and Promoter from Dendrobium officinale

作　　者：孙博 王义琴 何玲 王冬梅 黄鑫 师凯辉 陈宇 臧睿 和凤美[1] SUN Bo;WANG Yiqin;HE Ling;WANG Dongmei;HUANG Xin;SHI Kaihui;CHEN Yu;ZANG Rui;HE Fengmei(College of Horticulture and Landscape,Yunnan Agricultural University,Kunming,Yunnan 650201,China)

机构地区：[1]云南农业大学园林园艺学院,云南昆明650201

出　　处：《热带作物学报》2022年第9期1771-1778,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31760591);晋宁区花卉产业科技特派团(No.202204BI090022)

摘　　要：AGL6基因是MADS-box转录因子家族中的一员,是植物特有的转录调控因子,参与花器官形成,对唇瓣的形成起到关键作用。获得铁皮石斛DoAGL6基因及其启动子,并进行生物信息学分析,对DoAGL6基因启动子进行瞬时表达活性验证,可为进一步研究DoAGL6基因的功能提供参考。本研究克隆DoAGL6基因及其上游的启动子序列,利用在线软件对克隆得到的目的基因序列、启动子序列进行预测,分别构建由全长启动子和5'端缺失启动子驱动GUS的重组表达载体并瞬时转化拟南芥及铁皮石斛原球茎,探究其表达活性。结果表明,成功克隆了DoAGL6基因及其启动子,DoAGL6基因编码区长729 bp,编码243个氨基酸,编码蛋白分子式为C_(1213)H_(1955)N_(359)O_(375)S_(12),预测亚细胞定位于细胞核中;保守结构域分析表明,DoAGL6具有保守的MADS-box和K-box两个结构域,属于MADS基因家族MIKC亚家族。启动子序列长度为1891 bp,顺式作用元件分析结果显示,DoAGL6基因启动子中除了核心启动元件TATA-box、CAAT-box外,还有许多其他顺式作用元件,如脱落酸响应元件(ABRE)、光反应顺式调节元件(G-Box)、茉莉酸甲酯响应元件(TGACG-motif)、MADS-box蛋白结合位点(GArG-Box)等。成功构建融合表达载体pCAMBIA1300-A1-promoter::GUS、pCAMBIA1300-A2-promoter::GUS、pCAMBIA1300-A3-promoter::GUS,拟南芥和铁皮石斛原球茎瞬时转化及GUS组织化学染色结果表明,随着启动子5'端缺失片段的增长,GUS活性逐渐降低,启动子活性逐渐减弱,即全长启动子A1(-1891~1)的活性最强,5'端缺失片段A2(-1488~1)次之,A3(-784~1)活性最弱。瞬时转化后的拟南芥和铁皮石斛原球茎GUS染色均呈现蓝色,但与对照相比均较浅,说明全长启动子和2个5'端缺失启动子都具有驱动GUS的活性,但启动活性都比CaMV35S启动子的启动活性弱。As a member of MADS-Box transcription factor family,AGL6 gene is a plant-specific transcription regulator that participates in floral organ formation and plays a key role in labellum formation.DoAGL6 and its promoter were obtained from Dendrobium officinale,and bioinformatics analysis was conducted to verify the transient expression activity of DoAGL6 promoter,which can provide reference for further study on the function of DoAGL6.This study cloned DoAGL6 and its upstream promoter sequence,used online softwares for the purpose of the cloning gene and its promoter sequences to build respectively by the total length of the promoter and 5°-terminus of the recombinant expression vectors of promoter drive GUS and instantaneous transformation of Arabidopsis thalianaand D.officinale protocorm to explore its expression activity.The results showed that DoAGL6 and its promoter were cloned successfully.The coding region of DoAGL6 was 729 bp,encoding 243 amino acids.The molecular formula of the coding protein was C_(1213)H_(1955)N_(359)O_(375)S_(12),which was subcellular located in the nucleus.Conserved domain analysis showed that DoAGL6 had two conserved MADS-box and K-box domains,which belonged to MIKC subfamily of MADS gene family.The promoter sequence length was 1891 bp,cis-acting element analysis results showed that there were many other cis-acting elements in DoAGL6 gene promoter besides TATA-box and CAAT-box.Such as abscisic acid response element(ABRE),light response cis-regulatory element(G-box),methyl jasmonate response element(TGACG-Motif),MADS-box protein binding site(GARG-box)and so on.The fusion expression vectors pCAMBIA1300-A1-promoter::GUS、pCAMBIA1300-A2-promoter::GUS、pCAMBIA1300-A3-promoter::GUS were constructed successfully.GUS histochemical staining of A.thaliana and protocorms of D.officinaletransient transformation showed that with the growth of the 5°-terminal deletion fragments of the promoter,GUS activity gradually decreased,and promoter activity gradually decreased,that is,full-length promoter

关 键 词：铁皮石斛 AGL6基因 基因克隆 启动子 GUS活性 

分 类 号：S682.31[农业科学—观赏园艺]