滇龙胆GrWRKY5基因的克隆和植物表达载体构建

Cloning and construction of plant expression vector of GrWRKY5 gene in Gentiana rigescens

WRKY蛋白是目前被广泛研究的一类DNA特异结合转录因子,在植物次生代谢物生物合成、植物生长和发育及衰老等生理过程以及生物、非生物防御反应中起重要的调控作用。该研究从滇龙胆幼叶中克隆萜类合成的关键转录因子基因Gr WRKY5,并利用生物信息学方法对基因功能进行预测,构建植物过表达载体。结果表明:根据三年生滇龙胆转录组Gr WRKY5基因序列,设计特异性引物,通过RT-PCR扩增Gr WRKY5 ORF序列,并进行TA克隆、测序和序列分析;构建入门载体p ENTRTM2B-Gr WRKY5,经LR反应后构建植物过表达载体。Gr WRKY5 ORF全长591 bp,编码196个氨基酸,Gen Bank登录号为KF922375,其中第167与170之间的色氨酸(W)、精氨酸(R)、赖氨酸(K)、酪氨酸(Y)组成WRKY蛋白所特有的"WRKY"结构。序列分析表明Gr WRKY5是WRKY超家族的成员。经生物信息学在线软件分析发现,Gr WRKY5的等电点为6.29,脂肪族指数为61.37,不稳定指数为57.80。总平均疏水性为-0.708,为亲水蛋白;含有20种氨基酸,其中天冬氨酸(Asp)和脯氨酸(Pro)含量最高,为8.1%;半胱氨酸(Cys)含量最低,仅为1.0%。氨基酸序列系统发育分析表明,Gr WRKY5与拟南芥中WRKY家族中遗传距离最接近的是WRKY27,属于Ⅱe类成员;与Cr WRKY22和Vv WRKY22蛋白的亲缘关系较近;与Jc WRKY47和Tc WRKY27亲缘关系较远;BLASTp结果显示,Gr WRKY5与欧洲油菜Bn WRKY27-1的同源性最高(为69%);与拟南芥Aa WRKY22的一致性最低(仅为31%)。以Gateway入门载体p ENTR2B和目的载体p K2GW7为基础,成功构建了植物过表达载体p K2-35S-Gr WRKY5,该载体的成功构建为该基因在拟南芥、滇龙胆等植物中的遗传转化奠定了基础,同时为WRKY基因功能的深入研究提供依据。

WRKY protein is a class of specific DNA-binding transcription factor widely studied at present, which plays key regulating roles in the biosynthesis of plant secondary metabolites, the physiological processes of plant growth, development, senescence and the defense reactions of both biotic and abiotic stresses. To obtain the key transcription factors involved in the terpenoid biosynthesis,a gene GrWRKY5 was cloned from the spire of Gentiana rigescens. Its bioinformatic analysis was performed to predict its functions and its plant overexpression vector was also constructed. The gene specific primers were designed according to the transcript sequence of GrWRKY5 from the transcriptome of triennial G. rigescens. The open reading frame （ORF） of GrWRKY5 was obtained by Reverse Transcription Polymerase Chain Reaction （RT-PCR）. Then TA cloning, sequencing, and sequence analysis were performed. Entry vector pENTRTM2B-GrWRKY5 was constructed,and the plant overexpression vector of pKz-35S- GrWRKY5 was obtained after LR reaction. The ORF of GrWRKY5 has a length of 591 bp and encodes a predicated protein of 196 amino acids. The GenBank accession number for GrWRKY5 is KF922375. The tryptophan（W）,argi- nine（R）, lysine （K）, tyrosine （Y） between 167 and 170 in WRKY protein consist of the ＂ WRKY＂ symbolic structure. Sequence analysis showed that GrWRKY5 was a member of ＂WRKY＂ superfamily. Results of hioinforma tion software on line analysis showed that the theoretical isoelectric point （pI）,the aliphatic index and the instability index of GrWRKY5 protein were 6.29,61.37 and 57.80, separately. The GRAVY （grand average of hydropathicity） of GrWRKY5 protein was 0.708,which indicated that it belonged to hydrophilic protein. GrWRKY5 protein contained more than 20 kinds of amino acids,and the aspartic acid （Asp） and proline （Pro） accounted for the highest content （8.1%）,while Cysteine （Cys） content was the lowest accounting for only 1.0 %. Results of phylogenetic a nalysis showed that GrWRKY5 was at the same evolutionary branch with AtWRKY27, a member of class lie, and that GrWRKY5 was close to the CrWRKY22 protein of Catharanthus roseus and the VvWRKY22 protein of Vitis vinifera and was far from the JcWRKY47 protein in Jatropha curcas and the TcWRKY27 protein in Theobroma ca- cao. Results of BLASTp showed that GrWRKY5 protein had the highest identity （69G） with the BnWRKY27-1 protein in Brassica napus, while it had the lowest identity （31 % ） with the AaWRKY22 protein in Arabidopsis thaliana. The plant overexpression vector pKz-35S-GrWRKY5 which was suitable for agrobacterium tumefaciens and gene gun mediated transformation was successfully constructed base on entry vector pENTR2B and destination vector pK2GW7 of Gateway system. The successful construction of plant overexprossion vector pK2-35S-GrWRKY5 will provide a foundation for its genetic transformation into Arabidopsis thaliana and Gentiana rig escens and provide a basis for the further study of gene function.