小麦TaCRF2基因的克隆及其在烟草中的初步功能验证  

TaCRF2 Gene Isolation from Triticum aestivum and Primary Function Validation in Tobacco(Nicotiana tabacum L.)

作　　者：郭丽香[1,2] 高世庆[2] 唐益苗[2] 王永波[2] 刘美英[3] 张朝[3] 徐蓓[1,2] 赵昌平[2] 

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]北京市农林科学院杂交小麦工程技术研究中心,北京100097 [3]中国科学技术大学研究生院化学系,安徽合肥230026

出　　处：《作物学报》2011年第8期1389-1397,共9页Acta Agronomica Sinica

基　　金：国家转基因生物新品种培育重大专项(2008ZX08002-002);北京市自然科学基金项目(5102016);北京市科技新星计划项目(2007B056;2008B035);北京农林科学院院青年基金项目资助

摘　　要：通过RT-PCR方法从小麦cDNA中扩增获得1个锌指蛋白基因TaCRF2,该基因的cDNA长度为843bp,序列分析表明它编码1个含有280个氨基酸的蛋白质。在线软件预测该蛋白质的相对分子质量为30.97kD,等电点为7.03,且在C-末端含有1个典型的RING-H2型锌指蛋白结构域,在N-末端含有2个跨膜结构域。氨基酸序列比对发现,TaCRF2与水稻中1个RING型锌指蛋白(ABF95226)的相似度为82%。亚细胞定位分析显示,该蛋白分布在细胞核和细胞膜上。real-time RT-PCR表达特性分析显示,TaCRF2基因的表达受干旱、高盐和外源ABA的强烈诱导,低温对该基因的表达量影响不明显。初步功能验证发现过表达TaCRF2基因增强了转基因烟草对干旱和高盐胁迫的耐性。Wheat,like other crops, suffers from drought, salt, low-temperature and other abiotic stresses, seriously resulting in yield decline. It is an effective way for crop breeding to improve resistance via molecular biology techniques. Zinc finger protein, an important transcription factor commonly found in plant, can regulate the expression of multiple stress-inducible genes and enhance comprehensive resistances effectively. In this study, a RING-H2 zinc finger protein gene, designated as TaCRF2, was isolated from Triticum aestivum by RT-PCR. Its cDNA was 843 bp and encoded a putative protein of 280 amino acids with a predicted molecular mass of 30.97 kD and an isoelectric point （PI） of 7.03. A typical RING-H2 finger domain was found at the C-terminal region of TaCRF2 protein, and two transmembrane domains were found at the N-terminal region. Alignment of amino acid sequence showed that TaCRF2 was 82% identical to Oryza sativa putative RING zinc finger protein ABF95226. Subcellular localization analysis showed that the TaCRF2 was expressed in both nuclear and cytoplasm membrane. real-time PCR showed that the transcript of TaCRF2 was strongly induced by drought, salinity and cold to some extent. The TaCRF2 gene was transformed into tobacco cultivar Wisconsin 38 （W38） by Agrobacterium mediated under the control of the CaMV 35S promoter. Under drought, salt and low-temperature stresses, transgenic tobacco lines carrying TaCRF2 gene developed the strong primary root, more lateral root and smaller yellow leaves, and performed higher tolerance to these stresses than the wide-type tobaccos, The primary function verification showed that overexpression of TaCRF2 gene in tobaccos enhanced their tolerance to drought and salinity stresses. This study will help further research of wheat RING-H2 type zinc finger protein reactions in the role of stress resistance, and bring wheat resistance breeding excellent candidate genes.

关 键 词：烟草 小麦 RING-H2型锌指蛋白 TaCRF2基因 干旱胁迫 盐胁迫 

分 类 号：S572[农业科学—烟草工业]