Rht16矮秆小麦中GA2ox-A9(Rht18)基因的克隆与表达分析  

Cloning and Expression Analysis of GA2ox-A9(Rht18)Genes in Rht16 Dwarf Wheat

作　　者：林珊 魏淑红[1] 杨在君[1] 廖明莉[1] 陈真勇[1] 吴一超 Lin Shan;Wei Shuhong;Yang Zaijun;Liao Mingli;Chen Zhenyong;Wu Yichao(Key Laboratory of Southwest China Wildlife Resources Conservation,Ministry of Education,College of Life Sciences,China West Normal University,Nanchong,637009)

机构地区：[1]西华师范大学生命科学学院西南野生动植物资源保护教育部重点实验室,南充637009

出　　处：《分子植物育种》2023年第9期2796-2805,共10页Molecular Plant Breeding

基　　金：西华师范大学国家一般培育项目(19B039)资助。

摘　　要：赤霉素2-氧化酶(GA2ox)是赤霉素降解途径的一种关键酶,在茎的伸长中起着关键作用。为探讨GA2ox-A9(Rht18)与Rht16矮秆小麦矮化之间的关系,本研究利用同源克隆方法从硬粒小麦株高近等基因系ANW16F(含矮秆基因Rht16)和高秆材料LD222中分别分离得到GA2ox-A9-1和GA2ox-A9-2基因,并对其进行生物信息学分析和表达模式分析。结果表明,从矮秆ANW16F和高秆LD222克隆得到的GA2ox-A9-1和GA2ox-A9-2基因是位于A和B基因组的两个拷贝基因,均含有3个外显子和2个内含子,编码347个氨基酸。ANW16F和LD222的GA2ox-A9-1蛋白在第89位氨基酸残基存在差异,GA2ox-A9-2蛋白在第87位存在差异。ANW16F和LD222的GA2oxA9-1蛋白与GA2oxA9-2蛋白在第182、186、251、336位的4个氨基酸残基存在差异。GA2ox-A9为亲水性不稳定酸性蛋白,无跨膜结构,二级结构丰富,三级结构预测显示与拟南芥GA2ox 6ku3.1A蛋白的序列相似度高,GA2ox-A9-1蛋白结构完全相同,GA2ox-A9-2蛋白略有不同。系统进化分析显示,GA2ox-A9与野生二粒同源性最高。RT-qPCR结果显示,在拔节期GA2ox-A9在高秆LD222茎中的表达量显著高于ANW16F中的表达量,在孕穗期和抽穗期GA2ox-A9在矮秆ANW16F中的表达水平明显高于高秆LD222,推测GA2ox-A9(Rht18)基因参与了Rht16矮秆小麦的矮化。本研究为进一步探讨Rht16小麦的矮化机理和育种利用提供了理论依据。Gibberellin 2-oxidase(GA2ox)is a key enzyme in gibberellin degradation pathway that plays a key role in stem elongation.In this study,in order to explore the relationship between GA2ox-A9(Rht18)and dwarfing of Rht16 dwarf wheat,the GA2ox-A9(Rht18)gene are isolated by homologous cloning.Moreover,the bioinformatics analysis and expression pattern analysis are performed using the ANW16F(containing the dwarf gene Rht16)and tall LD222,a near-isogenic line of durum wheat as experimental materials.The results show that the GA2ox-A9-1 and GA2ox-A9-2 genes cloned from dwarf ANW16F and tall LD222 were two copies of genes located in the A and B genomes,which both contain three exons and two introns,encoding 347 amino acids.The GA2ox-A9-1 protein of ANW16F and LD222 differ at the amino acid residue at position 89 and GA2ox-A9-2 protein at position 87.The GA2ox-A9-1 and GA2ox-A9-2 proteins of ANW16F and LD222 differ from four amino acid residues at positions182,186,251,and 336.GA2ox-A9 is a hydrophilic unstable acidic protein with no transmembrane structure,and its secondary structure is rich.The prediction of tertiary structure reveals high similarity with the protein GA2ox6ku3.1A of Arabidopsis thaliana.The structures of GA2ox-A9-1 protein are identical,while the structures of GA2ox-A9-2 protein are slightly different.Phylogenetic analysis shows that GA2ox-A9 have the highest homology with wild emmer wheat.The RT-qPCR results show that the expression level of GA2ox-A9 in the stem of LD222 is significantly higher than that in ANW16F at jointing stage.At booting stage and heading stage,the expression level of GA2ox-A9 in ANW16F is significantly higher than that of LD222,suggesting that GA2ox-A9(Rht18)gene is involved in dwarfing of Rht16 dwarf wheat.This study provides a theoretical basis for further exploring the dwarfing mechanism and breeding and utilization of Rht16 wheat.

关 键 词：Rht16 GA2ox-A9基因 矮秆基因 生物信息学分析 实时荧光定量PCR 

分 类 号：S512.1[农业科学—作物学]