西瓜ClWRKY54基因的克隆、亚细胞定位及表达分析

Cloning, subcellular localization and expression analysis of Cl WRKY54 in Citrullus lanatus

WRKY转录因子是植物中所特有的一类转录因子,以基因家族形式存在,并在植物的生长发育和逆境响应等过程中发挥重要的作用。采用RT-PCR的方法从西瓜中分离得到一条西瓜CIRKY54基因。序列分析表明,该基因开放阅读框全长1 428 bp,编码475个氨基酸。其编码蛋白分子量约为51.82 KD,等电点为6.17。该基因蛋白序列中包含2个WRKY保守结构域,锌指结构为C2H2型,属于典型的1类WRKY基因。进化树分析显示,该基因蛋白序列同葫芦科作物中的甜瓜、黄瓜、西葫芦等的WRKY26具有较高的同源性,处于同一分支上。亚细胞定位分析显示,该基因编码蛋白定位于细胞核中。对该基因进行荧光定量PCR分析研究其表达特性,结果显示该基因在西瓜根、茎、叶中均有表达,但是在叶片中表达量最高;H2O2可以诱导该基因的表达,而乙烯处理可以抑制该基因的表达,这说明该基因可能参与逆境下H2O2和乙烯所介导的信号途径。在模拟干旱下,该基因表达无变化,说明该基因同干旱胁迫抗性不相关。本研究的结果将为进一步的解析ClWRKY54的功能奠定基础。

The WRKY transcription factors,uniquely existing in plants in the form of gene family,play important roles in plant growth,development and stress responses.In this study,a WRKY transcription factor gene CIWRKY54 was isolated from watermelon(Citrullus lanatus) leaves using reverse transcription PCR.Sequence analysis showed that the full length of the open reading frame of this gene was 1 428 bp,encoding 475 amino acids.The molecular weight of the encoded protein was about 51.82 KD and the isoelectric point was 6.17.The protein sequence of the gene contained two conserved WRKY domains with a C2 H2 type zinc finger structure,respectively,which was a typical feature of the WRKY Group 1.Evolutionary tree analysis showed that the protein sequence of the gene was highly homologous with WRKY26 of Cucurbitaceae crops such as melon,cucumber and zucchini.Subcellular localization analysis showed that the ClWRKY54 was located in the nucleus.The expression characteristics of the gene were analyzed by quantitative RT-PCR.Results showed that the gene was expressed in roots,stems and leaves of watermelon,but the highest expression was found in leaves.H2 O2 could induced the expression of CIWRKY54,while ethylene treatment could inhibit its expression,indicating that the gene might participate in the signal pathways mediated by hydrogen peroxide and ethylene under stresses.Under drought,the expression of CIWRKY54 remained unchanged,suggesting that it might not be related to drought stress.The results of this study would lay a foundation for further analysis of the functions of CIWRKY54.