摘要： 为研究水稻WRKY蛋白在应对各种生物以及非生物胁迫中发挥的作用，探明OsWRKY76是否参与调控水稻系统获得抗性，明确OsWRKY在植物抗病及耐逆反应过程中的作用机理，对OsWRKY76进行了氨基酸序列分析，发现OsWRKY76具有典型的WRKY结构域，属于WRKY-GCM1锌指WRKY超家族。克隆了OsWRKY76中300 bp的cDNA片段OsWRKY76-300，并扩增GUS基因中500 bp的片段作为连接物，通过片段末端人为增加的酶切位点将上述片段与Gateway系统的入门载体pENTR L16连接，形成中间由GUS 500连接、两端是OsWRKY76-300反向重复的发夹结构的载体，利用LR反应将dsWRKY76 pENTR L16中发夹结构的插入片段分别重组到诱导型双元载体GVG-TA7002和组成型双元载体Ubi-C1300中，获得RNA干扰载体。利用农杆菌介导的转化方法将诱导型双元表达载体转化水稻，获得7个转化株系。利用实时定量RT-qPCR检测转化株系中OsWRKY76的表达量，获得了2个沉默株系。

关键词: 水稻, WRKY转录因子, RNA干扰, 载体构建, 表达分析

Abstract: The WRKY protein family is the specific transcription factor family of plants, which is involved in plant growth, development and responses to various stresses. It is important to clarify the function of WRKY in biotic and abiotic stresses, and whether OsWRKY76 participates in regulating systemic acquired resistance in rice. Through analyzing the amino acid sequences, it was found that OsWRKY76 had a typical WRKY domain and belonged to the WRKY-GCM1 zinc finger WRKY superfamily. A 300 bp cDNA fragment of OsWRKY76, OsWRKY76-300, was cloned by PCR from the cDNAs of rice. A 500 bp fragment of GUS gene was amplified and was used as a linker. Both fragments were ligated into pENTR L16 vector, which was the entry vector in Gateway system, to form a hairpin vector in which two reverse repeated fragments of OsWRKY76-300 were linked by GUS 500 fragment. The inserted hairpin fragment was recombined into the inducible binary vector GVG-TA7002 and the constitutive binary vector Ubi-C1300, respectively, by LR reaction. And then the RNA interference vectors were obtained. Agrobacteria-mediated method was used to transform the inducible binary RNAi vector into rice. At last, seven transformed lines were got, and the expression level of OsWRKY76 was assayed by RT-qPCR. The expression of OsWRKY76 was silenced in two lines.

Key words: Rice, WRKY transcription factor, RNA interference, Vector construction, Expression analysis

中图分类号: 

• S511.03

• Q943.2