马铃薯StWRKY40基因的序列及表达模式分析

Sequence and Expression Pattern Analysis of Potato StWRKY40 Gene

WRKY转录因子是植物重要的调节因子,在植物逆境生长过程中起着关键的作用。为探究马铃薯WRKY转录因子在非生物胁迫下的调控作用,本研究以‘青薯9号’为试验材料,基于RT-PCR技术获取StWRKY40基因序列,利用DNAMAN、MEGA 7.0、ExPASy-ProtParam Tool等软件对其进行序列分析,通过QRT-PCR技术分析干旱胁迫(10%PEG-8 000)和盐胁迫(NaCl浓度为150 mmol/L)下该基因的表达模式。结果表明:StWRKY40的全长序列为1 713 bp,CDS序列为1 083 bp,该基因编码蛋白全长为360 aa,相对分子质量为39.79 kD,脂溶指数为64.72,理论等电点为8.37。其平均亲水系数为-0.698,说明StWRKY40基因编码的蛋白表现为亲水性。StWRKY40与番茄WRKY40的亲缘关系最近,相似性为94.16%。StWRKY40启动子除含有常见的CAAT-box顺式元件,还含有胁迫应答元件(TC-rich repeats)和干旱胁迫响应元件(DRE core)。QRT-PCR结果表明:盐胁迫4 d时,茎中的表达量最高,为对照的19.54倍;干旱胁迫8 d时,叶中的表达量最高,为对照的5.7倍。利用Gateway技术成功构建pJAM1502-StWRKY40表达载体。本研究为深入研究马铃薯WRKY基因的功能提供了理论基础,同时在提高马铃薯抗逆性能力和分子育种方面提供了一定的参考依据。

WRKY transcription factor is an important regulator of plants and plays a key role in the process of plant growth under adverse conditions. To explore potato WRKY transcription factors in regulating under abiotic stress, in this research, ’Qingshu9’ was used as the test material, based on RT-PCR technique to obtain StWRKY40 gene sequence, employing DNAMAN, MEGA 7.0, ExPASy-ProtParam tool software for bioinformatics analysis,applying QRT-PCR was used to analyze the expression pattern of this gene under drought stress(10% PEG-8 000)and salt stress(NaCl concentration was 150 mmol/L). The results show that the full-length sequence of StWRKY40 is 1 713 bp, the CDS sequence is 1 083 bp, the total length of the protein encoded by this gene was 360 aa, the relative molecular weight was 39.79 kD, the lipid solubility index was 64.72, and the theoretical isoelectric point was 8.37. The average hydrophilic coefficient was-0.698, indicating that the protein encoded by StWRKY40 gene was hydrophilic. StWRKY40 and tomato WRKY40 had the closest affinity with 94.16%. In addition to the common CAAT-box cis-elements, the StWRKY40 promoter contains stress response elements(TC-rich repeats) and drought stress response elements(DRE core). QRT-PCR results indicate that under 4 day of salt stress, the expression level in the stem was the highest, 19.54 times that of the control group. At 8 day of drought stress, the expression level in leaves was the highest, 5.7 times that of the control group. In summary, it can be preliminarily demonstrated that this gene is involved in the regulation of abiotic stress processes like drought and salt. The expression vector of pJAM1502-StWRKY40 was successfully constructed by Getway technology. This study provides a theoretical basis for in-depth study of the function of potato WRKY gene, and also produces a certain reference basis for improving potato stress resistance and molecular breeding.