小麦TaWRKY46-1基因克隆及建立在Gateway技术上的可诱导表达载体的构建  

Cloning of WheatTaWRKY46-1 Gene and Construction of Plant Inducible Expression Vector by Gateway Cloning Technique

作　　者：李明[1] 丁博[1] 牛有雄[1] 吕芳芳[1] 杨文丽[1] Silin Zhong 孙守钧[1] 谢晓东[1] 

机构地区：[1]天津农学院农学系,天津300384 [2]Boyce Thompson Institute for Plant Research, Cornell University, NY14853, USA

出　　处：《华北农学报》2011年第2期17-22,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家转基因新品种培育重大专项(2009ZX08009-084B)

摘　　要：以小麦品种扬麦158为材料,根据NCBI中小麦TaWRKY46基因序列设计引物,采用RT-PCR技术从小麦总RNA中分离了大小为870 bp的cDNA片段。测序表明,该片段与NCBI中已克隆的小麦TaWRKY46基因高度同源,包含完整的读码框,值得注意的是此蛋白保守氨基酸序列WRKYGQK突变为WRKYGEK,为TaWRKY46的新等位基因,命名为TaWRKY46-1。采用Gateway克隆技术构建了该基因的可诱导型超量表达载体,并转化农杆菌,为接下来转基因验证TaWRKY46-1的功能奠定了基础。In this study,the wheat variety,Yangmai 158（Triticum aestivem L.）,was used as the experimental material for cloning the wheat TaWRKY46 gene.Primers were designed based on a known wheat TaWRKY46 gene sequence in NCBI,subsequently an 870 bp cDNA fragment was amplified from total RNA of Yanmai 158 by RT-PCR.Sequence analysis revealed that the cloned fragment contains the fulL-length cDNA and is highly homologous to the TaWRKY46 sequence in NCBI.The sequence alignment revealed a Q to E change within the conserved ＂WRKYGQK＂ domain,suggesting it was a new allele of TaWRKY46.Hence we designated it as TaWRKY46-1.In order to elucidate the functional role of the new Yangmai 158 TaWRKY46-1 allele,a plant inducible over-expression vector,pKIGW-TaWRKY46-1,was constructed via Gateway cloning technology and transformed into Agrobacterium cells successfully.This work provides the key basis for future detailed functional analysis of wheat TaWRKY46-1 gene.

关 键 词：小麦 WRKY转录因子 Gateway克隆技术 可诱导表达载体 

分 类 号：Q785[生物学—分子生物学]