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

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c DNA,并对其进行序列分析。结果表明,该序列包含1个840bp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、TCrichrepeat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。

In order to understand the function of WRKY2 in zucchini(Cucurbita pepo),a length of 1071 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(1513 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.