‘云香’水仙NtWRKYY2基因的克隆及功能分析

Cloning and Functional Analysis of NtWRKYY2 Gene from Narcissus tazetta var.‘Yunxiang'

以‘云香’水仙为材料,采用PCR技术分离中国水仙WRKY转录因子家族成员NtWRKYY2(GenBank登录号为KX056496),其开放阅读框(ORF)长度为867bp,编码289个氨基酸。氨基酸序列比对及系统进化树分析显示,NtWRKYY2编码蛋白含1个WRKY结构域和C2H2锌指结构(Csx4Cx23HxH),属于第Ⅱd类WRKY转录因子。实时荧光定量PCR(qRT-PCR)分析显示,NtWRKYY2在‘云香’水仙应对盐胁迫中显著上调表达。利用InFusion克隆技术成功构建过表达载体pMDC140-NtWRKYY2,并采用农杆菌介导叶盘法转化烟草,获得11株卡那霉素抗性植株,转化子进一步PCR检测结果显示,其中有8株目的基因已成功导入烟草基因组中,转化率为72%。盐胁迫处理和叶绿素荧光参数分析显示,盐胁迫处理后NtWRKYY2过表达的转基因烟草萎蔫和黄化程度小于野生型植株,Fv/Fm值下降幅度小于野生型植株。研究表明,NtWRKYY2过表达的转基因烟草具有抵抗盐胁迫的能力。该研究为水仙抗盐转基因育种提供备选目的基因。

The WRKY transcription factors family gene NtWRKYY2（GenBank： KX056496）were cloned by PCR technology from Narcissus tazetta var.＇Yunxiang＇.The Open Reading Frame （ORF） of NtWRKYY2 contained 867 bp,encoding a protein of 289 amino acid residues.Multiple alignments and phylogenetic analysis showed that NtWRKYY2 proteins containing one WRKY consecutive structural domain and C2H2 type zinc finger（Csx4Cx23HxH）belong to Ⅱd sub-group of WRKY transcription factor together.Quantitative real-time PCR （qRT-PCR） analysis demonstrated that the NtWRKYY2 gene was obviously induced by salt stress in ＇Yunxiang＇.We constructed overexpressing vector pMDC140-NtWRKYY2 using In-Fusion cloning technique,and transformed into tobacco by the method of Agrobacterium though leaf disc transformation.The carrier of PCR results indicated that the resistant plantlets were positive and the converting rate of 72%.Salt stress and chlorophyll fluorescence parameter analysis showed that after the salt stress treatment,overexpression of NtWRKYY2 transgenic tobacco showed less wilt and chlorosis than the wild type plants,and the decrease of Fv/Fm value was less than the wild type plants.Studies have shown that overexpression of the NtWRKYY2 gene remarkably increased the salt tolerance in transgenic tobacco.This study offers an alternative gene for salt tolerant transgenic breeding of Narcissus.