青花菜谷胱甘肽S-转移酶的克隆及表达特性分析

Cloning and Expression Analysis of Glutathione S-transferases in Broccoli

本研究采用RT-PCR技术从青花菜自交系‘WN12-95B’中克隆到谷胱甘肽S-转移酶基因。序列分析表明,青花菜GST基因全长690 bp,ORF长度为675 bp,推导编码蛋白含有224个氨基酸,相对分子量为26.15 kD,理论pI值为6.56,属于Tau类家族成员。青花菜GST蛋白的二级结构主要以α-螺旋和延伸链为主。通过构建系统进化树发现,GST基因与油菜、芜菁亲缘关系最近。利用荧光定量PCR检测GST基因在青花菜保持系不同组织中的表达量,结果显示GST基因在根、叶、荚中的表达丰度较高,花蕾中表达丰度较低。青花菜Ogu不育系及其保持系不同发育阶段花蕾时空表达特性分析表明,花蕾发育早期(<2 mm)表达量最高,随着发育进程的推移,表达量逐渐下降。同时期不育系的表达量高于保持系。本研究为探讨青花菜GST基因在花粉发育过程中的功能提供一定理论依据。

In this study, the glutathione S-transferases gene was successfully cloned from a broccoli selfing line ＇WN12-95B＇ by RT-PCR technology. Sequence analysis revealed that the full length of GST gene in broccoli was 690 bp with an ORF of 675 bp, which encoded a polypeptide of 224 amino acids. The GST gene belonged to Tau type GST with the relative molecular weight of 26.15 kD and pI of 6.56. The secondary structure of GST protein in broccoli was mainly composed of α-helixs and extended chain. Through constructing phylogenetic tree, we found that the GST gene had the closest relationship with Brassiea napus and Brassiea rapa. qRT-PCR was used to detect the expression level of GST gene in different organs of broccoli maintenance line, the results of which indicated that GST gene had high expression level in root, leaf and silique but low level in bud. The analysis of time-space expression characteristics of buds at different development stages of Ogu CMS line and maintenance line showed that the expressive abundance of GST was the highest at early development stage of bud （〈2 mm）, and then the level decreased following the bud development. While the expression level of CMS line was higher than that of maintenance line at the same development stage of bud. This research could provide a theoretical basis for studying the function of GST gene in pollen development in broccoli.