西葫芦CpWRKY2基因的分离、鉴定及其对非生物胁迫分析

Isolation, Identification and Response to Abiotic Stress of CpWRKY2 Gene in Cucurbita pepo

摘要:

为了解西葫芦(Cucurbita pepo)的WRKY2的功能，通过转录组测序技术从叶片中分离到1条长度为1 071 bp的cDNA，并对其进行序列分析。结果表明，该序列包含1个840 bp的开放读码框，预测编码279个氨基酸，与中国南瓜(Cucurbita moschata，XM_023091218.1)的WRKY核苷酸序列相似性为98.51%，命名为CpWRKY2 (GenBank登录号: XM_023676898.1)。CpWRKY2定位于细胞核内，CpWRKY2蛋白包含有1个保守的WRKY结构域(第201~267位)，206~266位为WRKY蛋白DNA结合区域，锌指结构域(第232~264位)为C2H2型，且含有1个保守RTGHARFRRAP (第76~86位)氨基酸序列，属于典型的Ⅱd亚类WRKY家族蛋白。CpWRKY2基因上游启动子区域(ATG前的1 513 bp的序列)含有ARE、ABRE、MBS、TC-rich repeat和W-box等可能的胁迫响应顺式作用调控元件。CpWRKY2具有组织表达特异性，在花中表达量最高，其次为根和茎，在叶片和果实中的表达量较低。经5 ℃、10% PEG 6000模拟干旱胁迫，0.1 mmol/L ABA、50 mmol/L ETH、10 mmol/L SA和50 mmol/L MeJA处理后叶片中CpWRKY2均出现上调表达，因此，推测CpWRKY2基因参与了西葫芦不同非生物胁迫的防御反应。

Abstract:

In order to understand the function of WRKY2 in zucchini (Cucurbita pepo), a length of 1 071 bp cDNA, named CpWRKY2 (GenBank accession No. XM_023676898.1), was isolated from leaves by using transcriptome sequencing technology (i.e. RNA-seq). The cDNA contains an 840 bp open reading frame (ORF) encoding 279 amino acids. The nucleotide sequence of ORF was sharing over 98.51% with that from Cucurbita moschata (GenBank accession No.: XM_023091218.1), indicating a highly conservative evolution. CpWRKY2 protein was located in the nucleus, containing a conserved WRKY domain (position 201-267), a WRKY DNA binding domain (position 206-266), a zinc finger domain (position 232-264) of the C₂H₂ type, and a conserved RTGHARFRRAP (positions 76-86) amino acid sequence, which belonged to the typical IId subclass WRKY family proteins. The upstream promoter region of CpWRKY2 gene (1 513 bp before ATG) contained possible stress response cis-acting elements, such as ARE, ABRE, MBS, TC-rich repeat, and W-box. CpWRKY2 expression had tissue specificity, which was the highest in flowers, followed by roots and stems, and the lowest in leaves and fruits. Moreover, the expression of CpWRKY2 was overall up-regulated under stresses, such as 5 ℃, 10% PEG 6000, 0.1 mmol/L ABA, 50 mmol/L ETH, 10 mmol/L SA, and 50 mmol/L MeJA, suggesting CpWRKY2 gene might be involved in defense response to different abiotic stresses of C. pepo.