硬粒小麦赤霉素氧化酶基因GA2ox-A9的克隆及表达分析

Cloning and Expression Analysis of Gibberellin Oxidase Gene GA2ox-A9 in Durum Wheat

赤霉素2-氧化酶(GA2-oxidase,GA2ox)是赤霉素降解途径的关键酶,在茎的伸长中起着重要的作用。为探讨GA2ox-A9基因与小麦矮化之间的关系,本研究从硬粒小麦ANW16G(携带Reduced height gene18,Rht18)和其株高近等基因系LD222两个材料中克隆得到GA2ox-A9基因,分别命名为GA2ox-A9-1和GA2oxA9-2,并对其在不同发育时期的各个茎节进行了表达模式分析。序列分析显示,两个材料的GA2ox-A9基因均含有3个外显子和2个内含子,开放阅读框全长1044 bp,编码347个氨基酸,GA2ox-A9-1蛋白在第89位、275位、279位的氨基酸存在差异,GA2ox-A9-2蛋白在第182位、186位、251位、336位的氨基酸存在差异,编码的蛋白均具有2-酮戊二酸依赖性的双加氧酶典型的保守结构域。系统发育分析表明,硬粒小麦GA2ox-A9与其他物种具有较高的进化保守性,且与乌拉尔图最为接近。Real-time PCR结果显示,GA2ox-A9基因在矮秆小麦ANW16G中表达量最高,尤其是在拔节期茎中的表达量远远高于高秆对照LD222,表明与矮化性状有关。本研究结果为进一步研究GA2ox-A9基因的功能和小麦矮化的分子机制提供了理论依据。

GA2-oxidase(GA2 ox)is a key enzyme which catalyzes endogenous bioactive gibberellins formation and plays crucial roles in stem elongation.In order to explore the relationships between GA2 ox-A9 gene and dwarf wheat,GA2 ox-A9 genes,named GA2 ox-A9-1 and GA2 ox-A9-2,were cloned from durum wheat ANW16 G and its plant height near isogenic line LD222,respectively.The expression patterns of GA2 ox-A9 genes in different parts of stems at different developmental stages were analyzed.Sequence analysis showed that there were three exons and two introns in each of GA2 ox-A9-1 and GA2 ox-A9-1.Both had an open reading frame of 1044 bp,encoding347 amino acids.The deduced amino acid sequences of GA2 ox-A9-1 and GA2 ox-A9-2 had typical conserved domain of 2-ketoglutarate-dependent dioxygenase.There were differences in amino acids of GA2 ox-A9-1 protein in position 89,275,279 while GA2 ox-A9-2 protein in position 182,186,251 and 336.Phylogenetic analysis indicates that the GA2 ox-A9 showed high evolutionary conservation among durum wheat and other species,and the GA2 ox-A9 was most closed to Triticum urartu.The results of Real-time PCR showed that the expression of GA2 oxA9 gene was the highest in ANW16 G of dwarf wheat,especially in the stem at jointing stage,which was much higher than that of LD222,indicating that it was related to the dwarfing trait.The results provid a reference for further study on the role of GA2 ox-A9 gene in wheat dwarfing process.