摘要
糖基转移酶(glycosytransferase,GT)是催化活化的供体糖基转移到特异受体生成糖苷键的酶类,在应答多种生物和非生物胁迫中起重要作用。本研究利用PCR技术从陆地棉品种Coker 312基因组中分离克隆了GhGalT1基因的启动子,序列长度为539 bp,命名为pGhGalT1。启动子分析软件Plant CARE分析表明pGhGalT1含有CAAT-box、TATA-box核心元件,以及响应干旱、热、脱水、防御与胁迫应答的顺式作用元件MBS、HSE、MYCCONSE、TC-rich repeats和CGTCA-motif等。因此将pGhGalT1构建到启动子检测载体p BI101-GUS上,形成p BI101-pGhGalT1-GUS融合表达载体,以检测其启动子活性。通过农杆菌介导的浸花法转化拟南芥,经卡那霉素抗性筛选及PCR检测成功获得阳性转基因植株。对T3代转基因拟南芥进行组织化学染色分析显示该启动子主要在生长5~15 d的幼苗主根及侧根根尖表达,在子叶及莲座叶边缘也有微弱表达。非生物胁迫和激素处理后的组织化学染色、GUS酶活性及GUS基因定量分析结果显示GhGalT1基因的启动子受盐、渗透胁迫和激素(6-BA、MeJA、BL)的诱导,该结果为合理选用启动子改良作物提供理论依据。
Glycosytransferases(GTs)transfer an activated sugar donor to a specific acceptor to form glucosidic bond,which are regulated by various abiotic and biotic stresses,and may play a role in plant responses to changes in living conditions.In this study,a 539 bp fragment of GhGalT1 5′-flanking sequence was isolated from upland cotton Coker 312 by PCR,designated pGhGalT1.Analysis of pGhGalT1 sequence by PlantCARE revealed it contained not only putative CAAT box,TATA box sequence,but also MBS,HSE,TC-rich repeats,MYCCONSE and CGTCA-motif cis-acting element which involved in drought,heat,dehydration,defense and stress responsiveness.Thus,we constructed it into pBI101-GUS vector and formed pGhGalT1::GUS fusion expression vector(pBI101-pGhGalT1-GUS),then transferred the vector into Arabidopsis by the Agrobacterium-mediated floral dip method,and successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection.Histochemical assay of T3 generation of transgenic Arabidopsis revealed that GUS activities were mainly accumulated in root tips of primary and lateral roots in 5-to 15-day-old seedlings,and less strongly in cotyledons and rosette leaves.The histochemical staining results and the assay of quantitative GUS activity and GUS gene expression under abiotic stresses and hormone treatments revealed that the GhGalT1 promoter was salt-/osmotic-/6-BA-/MeJA-/BL-inducible.These findings contribute to the selection of a suitable promoter for crop molecular improvement.
作者
秦丽霞
李静
张换样
李盛
竹梦婕
焦改丽
吴慎杰
QIN Li-Xia;LI Jing;ZHANG Huan-Yang;LI Sheng;ZHU Meng-Jie;JIAO Gai-Li;WU Shen-Jie(Institute of Cotton,Shanxi Academy of Agricultural Sciences,Yuncheng 044000,Shanxi,China)
出处
《作物学报》
CAS
CSCD
北大核心
2018年第2期218-226,共9页
Acta Agronomica Sinica
基金
国家自然科学基金项目(31601350)
山西省基础研究项目(2015021152)
国家转基因生物新品种培育重大专项(2016ZX08005)资助~~
关键词
棉花
糖基转移酶
启动子
顺式作用元件
GUS组织化学染色
GUS定量分析
cotton(Gossypium hirsutum L.)
glycosytransferase
promoter
cis-acting element
GUS histochemical staining
quantitative GUS assay
