冷胁迫下甘蓝型冬油菜表达蛋白及BnGSTs基因家族的鉴定与分析  

Protein and physiological differences under cold stress,and identification and analysis of BnGSTs in Brassica napus L.

作　　者：马骊[1] 白静 赵玉红 孙柏林 侯献飞 方彦[1] 王旺田[1] 蒲媛媛[1] 刘丽君 徐佳 陶肖蕾 孙万仓[1] 武军艳[1] MA Li;BAI Jing;ZHAO Yu-Hong;SUN Bo-Lin;HOU Xian-Fei;FANG Yan;WANG Wang-Tian;PU Yuan-Yuan;LIU Li-Jun;XU Jia;TAO Xiao-Lei;SUN Wan-Cang;WU Jun-Yan(State Key Laboratory of Aridland Crop Science/Gansu Research Center of Rapeseed Engineering and Technology/College of Agronomy,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Zhangye Academy of Agricultural Sciences,Zhangye 734000,Gansu,China;Gansu Yasheng Agricultural Research Institute Co.Ltd,Lanzhou 730030,Gansu,China;Institute of Economic Crops,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,Xinjiang,China)

机构地区：[1]省部共建干旱生境作物学国家重点实验室/甘肃省油菜工程技术研究中心/甘肃农业大学农学院,甘肃兰州730070 [2]张掖市农业科学院,甘肃张掖734000 [3]甘肃亚盛农业研究院有限公司,甘肃兰州730030 [4]新疆农业科学院经济作物研究所,新疆乌鲁木齐830091

出　　处：《作物学报》2023年第1期153-166,共14页Acta Agronomica Sinica

基　　金：甘肃省青年科技计划项目(21JR7RA835);省部共建干旱生境作物学国家重点实验室(甘肃农业大学)开放基金课题(GSCS-2020-06);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-12)资助。

摘　　要：谷胱甘肽S-转移酶(glutathione S-transferases, GST)参与调节植物生长、发育和逆境胁迫反应的许多方面。本研究利用双向电泳和质谱技术分析‘16VHNTS309’在冷胁迫下差异表达的蛋白质,基于GO和KEGG分析鉴定出BE、APX、SOD、GST等参与冷胁迫反应的蛋白质。利用q RT-PCR和生理指标鉴定到响应冷胁迫的关键蛋白质GST,采用同源克隆法克隆到‘16VHNTS309’的GST基因。该基因CDS长度为642bp,编码213个氨基酸,是一个不稳定蛋白,属于GST_N_3谷胱甘肽S-转移酶家族,与甘蓝型油菜‘ZS11’序列相似性为99.22%,与‘ZS11’和‘Vision’两者的氨基酸序列相比较发现第127位亮氨酸(L)突变为脯氨酸(P)。基因家族分析表明,在甘蓝型油菜中共鉴定到153个Bn GSTs成员,按其功能主要分为Zeta、Phi、Theta、CHQ、DHAR、Lambda和Tau这七大类型,大部分的Bn GSTs属于Phi和Tau这2种类型。系统进化将Bn GSTs分为12个亚家族,亚家族Ⅰ和Ⅷ包含了较多的成员,Bn GSTs不均匀的分布在18条染色体上, C06染色体上分布Bn GSTs基因数量最多,含有10个保守的蛋白质基序。Bn GSTs基因家族中有99对基因存在共线性关系, 131个基因来自基因复制事件,片段重复事件在Bn GSTs基因的进化中起着重要作用。冷胁迫下Bna A02g35760D、Bna C06g20450D、Bna C06g35490D、Bna A02g03230D和Bna A02g35980D在强抗寒品种中的显著高表达,是弱抗寒品种的7~12倍,并且强抗寒品种具有较高的生理酶活性。另外,在冷冻胁迫下鉴定到一些瞬时和持续表达的关键候选基因。这些结果为进一步研究Bn GSTs基因在强抗寒甘蓝型冬油菜抗寒分子调控中的作用奠定基础。Glutathione S-transferases(GST) are involved in regulating many aspects of plant growth, development, and response to adversity stress. In this study, the differentially expressed proteins of ‘16VHNTS309’ under cold stress were analyzed using bidirectional electrophoresis and mass spectrometry, and proteins involved in the cold stress response, such as BE, APX, SOD, and GST, were identified based on GO and KEGG analyses. The key protein GST was identified using q RT-PCR and physiological indicators in response to cold stress, and the GST gene of ‘16VHNTS309’ was cloned using homologous cloning. This gene CDS length was 642 bp, and encoded 213 amino acids, which was an unstable protein and belonged to the GST_N_3 glutathione S-transferase family. The sequence similarity with Brassica napus ‘ZS11’ was 99.22%, and a comparison of the amino acid sequences of both ‘ZS11’ and ‘Vision’ revealed a mutation from leucine(L) to proline(P) at position 127. The gene family analysis showed that 153 Bn GSTs members were identified in Brassica napus and were classified into seven main types according to their functions: Zeta, Phi, Theta, CHQ, DHAR, Lambda, and Tau. Most Bn GSTs belonged to Phi and Tau types. Phylogeny divided Bn GSTs into 12 subfamilies, subfamilies Ⅰ and Ⅷ contained more members. Bn GSTs were unevenly distributed on 18 chromosomes, and the number of BNGSTs genes on C06 chromosome was the largest, it contained 10 conserved protein motifs. There were 99 pairs of genes in the Bn GSTs gene family that were colinearly related, 131 genes from gene duplication events, and segmental duplication events played an important role in the evolution of Bn GSTs genes. Significantly higher expression of Bna A02g35760D, Bna C06g20450D, Bna C06g35490D, Bna A02g03230D, and Bna A02g35980D in strong cold-resistant varieties was 7–12 times higher than that in weak cold-resistant varieties under cold stress. And the strong cold-resistant varieties had higher physiological enzyme activities. In addition, a num

关 键 词：甘蓝型冬油菜 冷胁迫 蛋白质谱 GST基因家族 表达模式 

分 类 号：S565.4[农业科学—作物学]