小麦泛素结合酶TaUBC16基因的克隆与功能分析

Cloning and functional analysis of ubiquitin-conjugating enzymes TaUBC16 gene in wheat

E2泛素结合酶在调控植物生长发育和胁迫信号转导过程中发挥着重要作用。本研究以小麦抗旱品种晋麦47的c DNA为模板克隆出E2泛素结合酶TaUBC16,该基因全长447 bp,编码148个氨基酸。顺式作用元件分析发现,TaUBC16启动子区含有与分生组织发育、胁迫响应、植物激素应答相关的多种顺式作用元件。利用小麦RNA-Seq转录组数据结合q RT-PCR验证分析发现,TaUBC16在小麦不同组织器官和发育阶段普遍表达,其中在30 d籽粒中的表达量较高,且均能被PEG-6000、甘露醇和ABA显著诱导表达。烟草叶片和小麦原生质体亚细胞定位分析表明,TaUBC16蛋白分布于细胞质和细胞核。通过异源表达TaUBC16转基因拟南芥进行生长发育表型分析发现,转基因株系开花时间早于野生型,其籽粒相比于野生型更为饱满,千粒重显著高于野生型。基于启动子区–388 bp位点(T-A)的多态性,开发了TaUBC16基因的竞争性等位基因特异性PCR (kompetitive allele-specific PCR,KASP)标记,鉴定了TaUBC16的单倍型,发现TaUBC16-Hap Ⅰ的千粒重、粒长和粒宽显著高于TaUBC16-Hap Ⅱ,并在我国小麦育种进程中得到正向选择。本研究结果将为进一步揭示TaUBC16基因参与调控小麦生长发育和响应逆境胁迫分子机理提供理论依据。

E2 ubiquitin-conjugating enzyme plays an important role in regulating plant growth and development,and stress signal transduction.In this study,the TaUBC16 gene encoding E2 ubiquitin-conjugating enzyme was cloned from the cDNA of the drought-tolerant wheat cultivar Jinmai 47.The gene was 447 bp in length and encoded 148 amino acids.The cis-acting element analysis showed that the promoter region of TaUBC16 contained various cis-acting elements related to meristem development,stress responses,and plant hormone responses.By the wheat RNA-seq transcriptome data analysis combined with qRT-PCR validation,it was found that TaUBC16 was generally expressed in different tissues/organs and at different growth stages of wheat,whereas the highest expression level was exhibited in developing grains at 30 days after anthesis.The relative expression level of TaUBC16 was highly induced by PEG,mannitol,and ABA stresses.The subcellular localization in tobacco leaves and wheat protoplasts showed that TaUBC16 proteins were located in both cytoplasm and nucleus.The phenotypic analysis of the heterologous expression of TaUBC16 in transgenic Arabidopsis revealed that the transgenic lines had earlier flowering time than the wild type,and its seeds were more plumpness with higher 1000-grain weight than the wild type.Based on the polymorphism of the promoter region-388 bp site(T-A),a kompetitive allele-specific PCR(KASP)marker of TaUBC16 gene was developed and its haplotypes were identified.The haplotype TaUBC16-Hap I had higher thousand-kernel weight,kernel length and width than TaUBC16-Hap II,and had been subjected to positive selection in wheat breeding processes in China.The results of this study provide a theoretical basis for further revealing the involvement of the TaUBC16 gene in the regulation of wheat growth and development and the molecular mechanism responding to adverse stresses.