紫花苜蓿MsZAT10基因的克隆及其在烟草中的功能验证  

Cloning and function analysis in tobacco of MsZAT10 from alfalfa

作　　者：孙亚男 林茹 潘晓阳 陈月[1] 陶磊[1] 郭长虹[1] SUN Ya-nan;LIN Ru;PAN Xiao-yang;CHEN Yue;TAO Lei;GUO Chang-hong(Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province,College of Life Science and Technology,Harbin Normal University,Harbin 150000,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与遗传育种重点实验室

出　　处：《草业学报》2019年第12期94-102,共9页Acta Prataculturae Sinica

基　　金：国家自然科学基金(31770575;31470571);国家高技术研究发展计划项目(863计划,2013AA102607);黑龙江省攻关项目(GA19B103);国家转基因生物新品种培育重大专项(2016ZX08004-002)资助

摘　　要：C2H2型锌指蛋白ZAT10在植物应对外界非生物胁迫中具有重要的作用。克隆得到紫花苜蓿C2H2型锌指蛋白MsZAT 10基因。该基因开放阅读框全长762 bp,编码253个氨基酸。ZAT10蛋白含有2个单C2H2型保守结构域,有典型的QALGGH保守基序,属于C2H2型锌指蛋白。氨基酸序列比对和进化树分析表明,紫花苜蓿MsZAT 10与蒺藜苜蓿MtZAT10亲缘关系最近,其氨基酸的相似性为76%。构建植物表达载体pCBM-MsZAT 10,通过农杆菌介导法转化到烟草,经过草丁膦(PPT)抗性筛选和聚合酶链式反应(PCR)鉴定,获得25株阳性植株。荧光定量PCR(qRT-PCR)检测表明,MsZAT 10基因在转基因株系中得到表达。选取3个转基因株系进行抗逆性分析,在-4℃条件下处理2 h,转MsZAT 10基因烟草叶片的萎蔫程度和相对电导率均低于野生型烟草。在0℃条件下处理5 h,转MsZAT 10基因烟草与野生型烟草相比积累更多的脯氨酸和可溶性蛋白,但丙二醛(MDA)含量要低于野生型烟草。另外,在300 mmol·L^-1 NaCl溶液中处理4 d,转MsZAT 10基因烟草的打孔叶圆片仍保持绿色而野生型烟草明显失绿。以上结果表明MsZAT 10基因在提高烟草对低温和盐胁迫的抗性方面具有重要的作用。C2H2 type zinc-finger proteins(ZFPs)play important roles in abiotic stress response in plants.In this study,a novel C2H2 ZFP family gene,MsZAT 10,from alfalfa(Medicago sativa),was cloned and characterized.The full length coding sequence(CDS)of MsZAT 10 is 762 bp,and encodes a protein of 253 amino acids containing two conserved single C2H2 domains and a typical QALGGH conserved motif.Phylogenetic analysis showed that MsZAT 10 shared sequence similarities of 76%with MtZAT10[the closet homologous gene in Medicago truncatula(XP_003592502.1)].The plant expression vector pCBM::MsZAT 10 was constructed and inserted into tobacco by Agrobacturium-mediated transformation.Twenty-five positive transgenic lines were obtained,verified from a phosphinothricin screen and PCR identification.The MsZAT 10 gene was expressed in transgenic lines confirmed by qRT-PCR analysis,and three transgenic lines were selected for further study.Transgenic tobacco plants showed higher tolerance and lower relative conductivity than those of wild type tobacco after exposure to a-4℃ temperature for 2 h.The tissue proline and soluble protein concentrations in transgenic tobacco were higher than those in wild type tobacco exposed to 0℃ for 5 h.In contrast,malondialdehyde levels were lower than those in wild type tobacco.After treatment with 300 mmol·L^-1 NaCl for 4 days,the leaf discs remained green in transgenic tobacco while the wild type tobacco turned chlorotic.These results indicated that overexpression of MsZAT 10 enhanced plant tolerance to low temperature and salt stress in transgenic tobacco.

关 键 词：紫花苜蓿 MsZAT 10基因 低温胁迫 盐胁迫 功能 

分 类 号：S54[农业科学—作物学]