大豆GmCBL7基因的克隆及生理功能分析  

Cloning and Physiological Function Analysis of GmCBL7 Gene in Soybean(Glycine max)

作　　者：陈男 李静[1] 沈祥娟 彭亚男 孙丹丹 王全伟[1] CHEN Nan;LI Jing;SHEN Xiang-Juan;PENG Ya-Nan;SUN Dan-Dan;WANG Quan-Wei(College of Life Science and Technology,Harbin Normal University/Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province,Harbin 150025,China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院/黑龙江省分子细胞遗传与遗传育种重点实验室,哈尔滨150025

出　　处：《农业生物技术学报》2021年第10期1904-1913,共10页Journal of Agricultural Biotechnology

基　　金：哈尔滨市科技创新人才研究专项(RC2013,QN002103);黑龙江省博士后资助项目(LBH-Z13185);黑龙江省教育厅科学技术研究项目(12511154);哈尔滨师范大学博士科研启动基金(KGB200903)。

摘　　要：大豆(Glycine max)是我国主要的粮食和油料作物,盐碱和干旱等逆境会对大豆的产量与品质产生严重影响。钙调磷酸酶B样蛋白(calcineurin B-like protein, CBL)是一类钙离子受体,在植物逆境胁迫响应中起重要作用。本课题组前期对大豆CBL基因家族表达模式分析的结果表明,大豆GmCBL7可被高盐和干旱诱导表达。为进一步研究GmCBL7基因与耐盐性和抗旱性的关系,本研究利用反转录PCR(reverse transcription-PCR, RT-PCR)技术从大豆中克隆得到了GmCBL7 (GenBank No. XM 006578843.3)基因。该基因CDS全长672 bp,编码223个氨基酸,蛋白质的分子量为25.8 kD,理论等电点为4.81,属酸性蛋白,具有典型的EF-hand结构域,无信号肽,是非分泌性蛋白。分析系统进化表明,GmCBL7与豆科植物中的同源蛋白处于同一个进化分支,且与野生大豆(Clycine soja)中同源蛋白的亲缘关系最近。构建植物表达载体pCPB-GmCBL7对烟草(Nicotiana tabacum)进行遗传转化,并对转GmCBL7基因烟草的抗逆性生理功能进行分析,结果表明,在高盐(200 mmol/L NaCl)和干旱(20%PEG6000)胁迫处理6和12 h后,转基因烟草植株的超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)和过氧化物酶(peroxidase, POD)活性和脯氨酸含量均显著高于野生型(P<0.05),丙二醛(malondialdehyde, MDA)含量显著低于野生型(P<0.05),说明过表达GmCBL7基因能够提高转基因烟草的耐盐性和抗旱性。本研究为进一步探讨GmCBL7基因的功能提供了依据,并为大豆抗逆育种提供了新的基因。Soybean(Glycine max) is main grain and oil crops in China, salinity and drought have seriously affected the yield and quality of soybeans. Calcineurin B-like protein(CBL) is a type of calcium receptor,which plays an important role in the process of plant stress response. The results of the expression pattern analysis of soybean CBL gene family in the early stage of this research group showed that soybean GmCBL7 could be induced by high salt and drought.In order to further study the relationship between GmCBL7 gene and salt tolerance and drought resistance. In this study, GmCBL7(GenBank No. XM 006578843.3) was cloned from soybean by reverse transcription-PCR(RT-PCR) technology. The CDS sequence of the gene was 672 bp in length, encoding 223 amino acids, the molecular weight of the protein was 25.8 kD, and the theoretical isoelectric point was 4.81. It was an acidic protein with a typical EF-hand domain and no signal peptide. It was a non-secreted protein. Analysis of systematic evolution showed that GmCBL7 was in the same evolutionary branch as the homologous protein in legumes, and had the closest relationship with the homologous protein in wild soybean(Clycine soja). The plant expression vector pCPB-GmCBL7 was constructed to genetically transform tobacco(Nicotiana tabacum), and analyzed the physiological functions of the GmCBL7 transgenic tobacco for stress resistance, the results showed that after high salt(200 mmol/L NaCl) and drought(20%PEG6000) stress treatments for 6 and 12 h, the activity of superoxide dismutase(SOD), catalase(CAT) and peroxidase(POD) and content of proline were significantly higher than those of wild type, while the content of malondialdehyde(MDA) was significantly lower than that of wild type. It was suggested that overexpression of GmCBL7 gene could improve salt tolerance and drought resistance of transgenic tobacco.This study provides a basis for further exploring the function of GmCBL7 gene, and provides a new gene for soybean salt and drought resistance breeding.

关 键 词：大豆 大豆钙调磷酸酶B样蛋白(GmCBL7)基因 生物信息学分析 表达分析 功能分析 

分 类 号：S565.1[农业科学—作物学]