陆地棉胆色素原脱氨酶基因的克隆及表达分析  

Cloning and Expression Analysis of Porphobilinogen Deaminase Genes in Upland Cotton(Gossypium hirsutum)

作　　者：陈义珍 李浩[1] 傅明川 王立国[1] 刘任重[1] 柳展基[1] CHEN Yi-Zhen;LI Hao;FU Ming-Chuan;WANG Li-Guo;LIU Ren-Zhong;LIU Zhan-Ji(Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain,Ministry of Agriculture/Shandong Cotton Research Center,Jinan 250100,China)

机构地区：[1]农业部黄淮海棉花遗传改良与栽培生理重点实验室/山东棉花研究中心,济南250100

出　　处：《农业生物技术学报》2020年第7期1221-1229,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31901422);山东省农业科学院农业科技创新工程项目(CXGC2018E06)。

摘　　要：叶色突变体是研究高等植物叶绿素代谢、叶绿体发育、光合作用和激素生理等一系列生理代谢过程的理想材料,也是遗传和育种研究中的重要材料。胆色素原脱氨酶(porphobilinogen deaminase,PBGD)是植物叶绿素生物合成的关键酶。本研究以陆地棉(Gossypium hirsutum)'鲁棉418'的花斑叶突变体为材料,克隆了4个GhPBGDs,分别命名为GhPBGD1A(GenBank No.MN849924)、GhPBGD1D(GenBank No.MN849925)、GhPBGD2A(GenBank No.MN849926)和GhPBGD2D(GenBank No.MN849927)。测序结果表明,正常叶片与花斑叶片中的4个基因序列相同;4个基因均含有5个外显子和4个内含子,预测结果均定位于叶绿体;进化分析表明,GhPBGD蛋白与拟南芥(Arabidopsis thaliana)和油菜(Brassica napus)的PBGD同源蛋白亲缘关系较近。另外,4个基因启动子顺式作用元件中含有光响应元件、厌氧环境及多种激素相关的应答元件。qRT-PCR结果表明,4个GhPBGD基因在正常棉株的叶、花、铃和苞叶中均有表达,其中在叶片中的表达量最高;与正常叶相比,花斑叶中GhPBGD基因的表达量都显著降低。以上结果表明,GhPBGD基因家族可能参与调控叶色突变。本研究可为深入探讨叶色缺失突变体的分子机理提供参考依据。Leaf color mutants are ideal materials for studying a series of physiological metabolic processes such as chlorophyll metabolism,chloroplast development,photosynthesis,and hormone physiology in higher plants,as well as important materials in genetics and breeding researches.Porphobilinogen deaminase(PBGD)is the key enzyme in the process of plant chlorophyll biosynthesis.In this study,4 PBGD genes were isolated using mottled leaf mutant of upland cotton(Gossypium hirsutum)'Lumian 418',and named as GhPBGD1A(GenBank No.MN849924),GhPBGD1D(GenBank No.MN849925),GhPBGD2A(GenBank No.MN849926)and GhPBGD2D(GenBank No.MN849927),respectively.The sequence analysis showed that each of the 4 GhPBGD genes had no difference in sequences between the normal and mottled leaves.All GhPBGD genes had a similar gene structure with 5 exons and 4 introns,and were predicted to be located in chloroplasts.Phylogenetic analysis indicated that GhPBGDs had close relationship with the PBGD proteins from Arabidopsis thaliana and Brassica napus.In addition,the cis-elements analysis revealed that many elements involved in light,anaerobic and hormone responsiveness were identified in the promoter regions of GhPBGD genes.Furthermore,qRT-PCR analysis showed that GhPBGD genes expressed in leaves,flowers,bolls and sepals,and had the highest expression in the leaves.Compared to normal leaves,the expression of GhPBGD genes were significantly down-regulated in the mottled leaves.The above results suggested that GhPBGD genes might be involved in the regulation of leaf color mutation.This study could provide reference data for further exploration of the molecular mechanism of leaf color deletion mutants.

关 键 词：棉花 胆色素原脱氨酶(PBGD) 基因克隆 表达分析 

分 类 号：S562[农业科学—作物学]