浙贝母GA20ox、NCED、IAA的克隆、表达及在鳞茎发育中的作用  

Cloning and Expression of GA20ox,NCED and IAA and Their Roles in Bulb Development of Fritillaria thunbergii

作　　者：李梓铭 范小平 金泽兰 江建铭[2] 王志安[2] 王忠华[1] LI Zi-Ming;FAN Xiao-Ping;JIN Ze-Lan;JIANG Jian-Ming;WANG Zhi-An;WANG Zhong-Hua(Institute of Biotechnology,Zhejiang Wanli University,Ningbo 315100,China;Zhejiang Institute of Traditional Chinese Medicine,Hangzhou 310023,China)

机构地区：[1]浙江万里学院生物技术研究所,宁波315100 [2]浙江省中药研究所,杭州310023

出　　处：《农业生物技术学报》2021年第9期1710-1721,共12页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划项目(2017YFC1700700);现代农业产业技术体系建设专项(CARS-21);浙江省中药材新品种选育重大科技专项(2016C02058);浙江省重点实验室建设项目(2011E10015);浙江省重点研发计划项目(2017C02011);宁波市公益项目(2019C10011)。

摘　　要：赤霉素(gibberellin,GA)、脱落酸(abscisic acid,ABA)和生长素(auxin,IAA)作为植物体内的内源调节激素,对植物生长发育以及植物对逆境胁迫的响应起着重要作用。赤霉素双加氧酶(gibberellin 20-oxidase,GA20ox)是赤霉素合成途径最后阶段的关键限速酶;9-顺式环氧类胡萝卜素双加氧酶(9-cisepoxycarotenoid dioxygenase,NCED),是脱落酸间接合成途径的关键限速酶;生长素应答蛋白(auxin/indole-3-acetic acid,AUX/IAA)可负调控IAA的合成。本研究使用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)测定了浙贝母(Fritillaria thunbergii)鳞茎发育过程中GA、ABA、IAA的含量变化。结果显示,随着鳞茎发育,GA含量呈下降趋势,ABA含量呈上升趋势,IAA含量大致呈下降趋势。采用同源克隆结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术成功克隆了浙贝母的基因GA20ox(GenBank No.MW238816)、NCED(GenBank No.MW238817)和IAA(GenBank No.MW238818);生物信息学分析显示,GA20ox基因cDNA序列全长1490 bp,开放阅读框为1122 bp,编码373个氨基酸;NCED基因cDNA序列全长2270 bp,开放阅读框为1800 bp,编码599个氨基酸;IAA基因cDNA序列全长1255 bp,开放阅读框为864 bp,编码287个氨基酸。qRT-PCR分析表明,GA20ox、NCED和IAA的组织特异性表达类似,在根、茎、叶中的表达量均高于鳞茎中的;不同发育时期鳞茎的GA20ox、NCED、IAA基因表达量与GA、ABA、IAA的相关性分析显示GA20ox、NCED与GA、ABA含量呈极显著正相关(P<0.01),IAA与IAA的含量呈极显著负相关(P<0.01)。本研究对GA20ox、NCED、IAA基因在GA、ABA、IAA代谢中的功能研究提供了理论依据,为进一步探究GA20ox、NCED、IAA基因在浙贝母鳞茎发育过程的分子机制提供参考。Gibberellin(GA)is a type of diterpene plant hormone,which plays an important role in the physiological development of plants.GA can promote the plant extension and affect the production of dry matter in sink organs.It can also activateα-amylase activity to promote catalyzes the degradation of energy storage substances and promotes the break of plant dormancy.For example,the increase of GA content can promote the germination of barley and make it enter the development stage earlier.GA also has an effect on fruit development and maturity,it can also delay leaf senescence and inhibit tuber formation.Gibberellin 20-oxidase(GA20ox)enzyme is the key rate-limiting enzyme in the final stage of GA synthesis pathway.It exercises catalytic regulation function in the final stage of GA synthesis and promotes the production of GA20 and GA9,those are precursors of active GA1 and GA4.GA20ox gene has a close influence on the activity of GA20ox enzyme and is closely related to GA.ABA(abscisic acid)is a 15-carbon sesquienene compound,which can effectively promote the development and growth of various plants.It plays an important role in seed germination,maturation and dormancy,fruit maturation.It can bring huge economic benefits and can effectively increase crop yields.There are 2 ways to synthesize ABA in plants,one is the indirect pathway,the C15 pathway,and the other is the direct pathway,which also can be called the carotenoid pathway.The synthesis of ABA in higher plant tissues is mainly achieved through indirect pathways.9-cis epoxy carotenoid dioxygenase(NCED)is the rate-limiting enzyme in the process of indirect ABA synthesis and regulation.Auxin(IAA)is an endogenous hormone containing an unsaturated aromatic ring and an acetic acid side chain.IAA is a signal transduction compound that promotes and affects plant development and physiological changes.It plays an important role at every moment of development,affecting the division,differentiation and elongation of plant cells,as well as various physiological activities of pl

关 键 词：浙贝母 鳞茎发育 内源激素 基因克隆 基因表达 

分 类 号：S567.231[农业科学—中草药栽培]