亚洲棉油菜素内酯合成酶基因(GaCPD2)的克隆、表达分析及功能验证  

Cloning, Expression Analysis and Functional Verification of Brassinosteroids Biosynthese Gene(GaCPD2) in Cotton(Gossypium arboreum L.)

作　　者：鲁丽丽[1,2] 屈凌波[1] 杨作仁[2,3] 杨召恩[2,3] 郑武[2,3] 葛晓阳[2,3] 李付广[2,3] 

机构地区：[1]河南工业大学,郑州450001 [2]棉花生物学国家重点实验室,安阳455000 [3]中国农业科学院棉花研究所,安阳455000

出　　处：《农业生物技术学报》2014年第7期805-815,共11页Journal of Agricultural Biotechnology

基　　金：国家自然基金-新疆联合基金重点项目(No.U1303282);国家棉花产业技术体系-分子育种(No.CARS-18-02)

摘　　要：持续光形态建成与矮化基因(constitutive photomorphogenesis and dwarf,CPD)是油菜素内酯(brassinosteroids,BRs)合成中的关键限速酶,为研究棉花中CPD基因表达模式和验证其功能,采用RTPCR方法首次从亚洲棉(Gossypium arboreum L.)石系亚1号中克隆到了一个细胞色素P450单加氧化酶基因GaCPD2(GenBank登录号:KJ183066)。生物信息学分析表明,该基因CDS序列长度1 413 bp,编码470个氨基酸,理论相对分子质量53.98 kD,理论等电点9.40,主要由α-螺旋、β-折叠、延伸链和无规则卷曲组成。多序列比对结果表明,GaCPD2具有细胞色素P450保守结构域,氨基酸序列与其他已报道物种中同源基因的相似性最高为84.0%。荧光定量PCR结果表明,GaCPD2在根、茎、叶、花和纤维组织中均有表达,开花后第10天的纤维中表达量最高,是0DPA的10倍左右。克隆了GaCPD2基因启动子序列,生物信息分析表明,GaCPD2基因启动子包含大量与光响应有关的顺式作用元件以及部分胁迫应答元件,推测其可能受光调控,参与胁迫应答。构建过表达载体转化拟南芥油菜素内酯合成突变体cpd91(Columbia背景),发现转基因株系的叶片展平,株高及育性恢复至野生型,表明GaCPD2基因在BR合成中起着重要作用,为研究油菜素内酯对棉花生长发育的影响提供了基础资料,有助于揭示BR调控植物株型及产量性状的分子机理。Constitutive photomorphogenesis and dwarf gene （CPD） is a key rate-limiting enzyme in brassinosteroids（BRs） synthesis,gene expression patterns and functions of which in Arabidopsis,rice （Oryza sativa） and tomato （Lycopersicon esculentum） have been researched,and not in cotton（Gossypium arboreum L.）.To clone and verify CPD gene function in cotton,a cytochrome P450 monooxygenase gene,GaCPD2 （GenBank accession：KJ183066） was cloned from cotton by RT-PCR.Expression pattern and functional bioinformatics analysis showed that GaCPD2 was 1 413 bp,which encoded a deduced protein including 470 amino acid residues with theoretical relative molecular weight of 53.98 kD and pI 9.40,mainly composed of alpha helix,beta turn,extended strand and randon coil.Multiple sequence alignment revealed that the most similarity was 84.0％ between the amino acid sequence of GaCPD2 and that of other reported species,which had conserved domain of cytochrome P450.Real-time PCR revealed that GaCPD2 expressed in root,stem,leaf,flower and fiber,and had the highest expression level in 10 days post-anthesis （DPA）fiber,which was about 10 times that of 0DPA.Cloned promoter sequence of GaCPD2 contained many cis-elements related to light response by bioinformatic analysis,which implied that GaCPD2 was light regulated.Overexpression vector 35S：：GaCPD2 for GaCPD2 transformation restored the growth of cpd91,which is a brassinosteroids deficient dwarf mutant of Arabidopsis （Columbia background）.The transgenic lines showed flattened leaves and restored fertility.These results revealed that GaCPD2 played an important role in brassinosteroids biosynthese.These results lay the base for the further reseach of BR regulation in cotton development,and will help to reveal the BR regulation of plant molecular mechanism of plant type and yield traits.

关 键 词：亚洲棉 GaCPD2 油菜素内酯 表达分析 功能验证 

分 类 号：S482.8[农业科学—农药学]