马铃薯WRKY2基因的克隆和非生物逆境下的表达模式

Cloning and expression pattern research under abiotic stress of the WRKY2 in potato

关键词:  马铃薯  WRKY  系统发育  EMSA  表达模式

Key words:  ession pattern

摘要:

马铃薯WRKY2基因的功能尚未见有报道,WRKY蛋白是近年来发现的一类重要转录因子家族,它们在植物应对生物胁迫、非生物胁迫和生长发育过程中起到关键的调控作用。该研究采用电子克隆法获得马铃薯WRKY2基因,该基因的编码区长度1 065 bp,编码355氨基酸,序列分析表明,该蛋白属于WRKY家族第二组成员,锌指结构为C-X₅-C-X₂₃H-X-H。构建系统发育树结果表明它与番茄WRKY7亲缘关系较近,氨基酸序列相似性达96%,与烟草中WRKY蛋白的相似性为86%,利用原核表达法在大肠杆菌中获得该蛋白。通过凝胶阻滞实验证明,该蛋白在体外能结合W-box元件,而且这种结合能被冷探针所竞争,同时也表明StWRKY2不能结合含有突变W-box DNA片段,证明StWRKY2与W-box结合具有特异性。通过实时荧光定量PCR技术分析该基因在根、茎和叶中的表达量,结果表明该基因主要在根中表达,其次是叶和茎。为进一步研究该基因可能参与的生理功能,对马铃薯组培苗进行10 μmol/L 低磷、10 μmol/L低钾、200 mmol/L NaCl、400 mmol/L PEG溶液和4 ℃低温处理,处理时间6 h,实时荧光定量PCR的结果表明该基因在低磷处理后表达量明显下降,在200 mmol/L NaCl和400 mmol/L PEG处理6 h后表达量明显升高,但在10 μmol/L低钾和4 ℃低温处理后表达量与对照相比无明显变化。说明StWRKY2能响应低磷、NaCl和PEG这三种非生物逆境胁迫。研究结果可为进一步深入研究马铃薯WRKY2基因的功能奠定基础。

Abstract:

Function of StWRKY2 has not been reported. Plant WRKY proteins are found as an important class of transcription factors in recent years, which are widely involved in biotic stress, abiotic stress and growth development regulation, and they are mainly divided into three groups. WRKY2 was cloned from potato by homology cloning, with length of the coding region 1 065 bp,encoding 355 amino acids by DNA sequencing. With the amino acid sequence of StWRKY2 blasting in NCBI, 19 homology amino acid sequences were selected to analysis conservative domain. Amino acid analysis showed that the StWRKY2 contained 1 conserved WRKY domain(TTGACC)and belonged to the second group of WRKY family members, and its zinc-finger structure was C-X₅-C-X₂₃H-X-H. Phylogeny tree results showed StWRKY2 was the most closest to SlWRKY7(Solanum lycopersicum)with 96% similarity, and the similarity of a WRKY protein of tobacco was 86%. The protein-GST(glutathione-S-transferase)complex was obtained in Escherichia coli by using the method of prokaryotic expression. StWRKY2-GST could combine W-box in vivo by Electrophoretic Mobility Shift Assay analysis,but only GST protein could not combine W-box. StWRKY2-GST combination with W-box could be competed by cold probe(unlabeled probe). And the experiment results also showed that StWRKY2-GST could not combine mutation W-box,this suggested StWRKY2-GST combined W-box with specificity. Analysis of StWRKY2 expression level in the root, stem and leaf tissue through the real-time fluorescence quantitative PCR,the results showed that the gene was mainly expressed in the root. Next was leaf and stem. In order to further study the possible function of the gene,potato seeding from tissue culture were treated under 10 μmol/L low phosphorus,10 μmol/L low potassium,200 mmol/L NaCl,400 mmol/L PEG solution and 4 ℃ low-temperature treatment for 6 h,real-time fluorescence quantitative PCR showed that this gene expression level decreased significantly after low phosphorus treatment, expression of StWRKY2 increased significantly after 200 mmol/L NaCl and 400 mmol/L treatment. Expression level of StWRKY2 had no obvious change after low potassium and 4 ℃ low temperature treatment. The results indicated that StWRKY2 could in response to low phosphorus,NaCl and PEG three kinds of abiotic stresses. These results would lay a solid foundation for further study of potato WRKY2 gene function.