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陆地棉转录因子GhWRKY91基因的原核表达及鉴定
顾丽姣,魏恒玲,喻树迅
0
(中国农业科学院棉花研究所,棉花生物学国家重点实验室,安阳 455000; 河北农业大学林学院,保定 071000)
摘要:
为了筛选出高表达棉花GhWRKY91可溶性蛋白的原核表达载体。以中棉所10号叶片的cDNA为模板PCR扩增GhWRKY91基因,扩增产物分别构建到4个不同的原核表达载体pET-22b(+)、pET-32a(+)、pMAL-c5x和pGEX-4T-1。将重组载体pET-22b(+)-GhWRKY91、pET-32a(+)-GhWRKY91、pMAL-c5x-GhWRKY91和pGEX-4T-1-GhWRKY91转化到大肠杆菌菌株BL21(DE3)或Arctic-Express™(DE3)RP中,IPTG诱导表达,SDS-PAGE电泳分析不同原核表达载体的蛋白表达情况。结果显示,pET-22b(+)-GhWRKY91 和pET-32a(+)-GhWRKY91在BL21(DE3)中以及pMAL-c5x-GhWRKY91在Arctic-Express™(DE3)RP中均未见明显蛋白表达,而pGEX-4T-1- GhWRKY91在Arctic-Express™(DE3)RP中表达的蛋白基本存在于上清中,可获得可溶性蛋白。因此,将GhWRKY91基因构建到pGEX-4T-1原核表达载体上可成功获得可溶性蛋白。
关键词:  棉花  GhWRKY91基因  SDS-PAGE  原核表达  表达分析
DOI:10.13610/j.cnki.1672-352x.20220106.012
基金项目:国家棉花产业体系(CARS-15-06)和河北农业大学引进人才科研专项(YJ2021011)共同资助。
Prokaryotic expression and identification analysis of transcription factor GhWRKY91 gene in cotton (Gossypium hirsutum)
GU Lijiao,WEI Hengling,YU Shuxun
(State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000; College of Forestry, Hebei Agricultural University, Baoding 071000)
Abstract:
The study was aimed at screening out the prokaryotic expression vector with high expression of cotton GhWRKY91 soluble protein. The GhWRKY91 gene was amplified by PCR using cDNA of CCRI10 leaves as template, and the amplified products were constructed into four different prokaryotic expression vectors, pET-22b(+), pET-32a(+), pMAL-c5x and pGEX-4T-1, respectively. The recombinant vectors, pET-22b(+)- GhWRKY91, pET-32a(+)-GhWRKY91, pMAL-c5x-GhWRKY91 and pGEX-4T-1-GhWRKY91 were transformed into Escherichia coli strains BL21(DE3) or Arctic-Express™(DE3)RP. The fusion protein expression was induced by IPTG, and SDS-PAGE electrophoresis was used to analyze the protein expression by different prokaryotic expression vectors. The results showed that no obvious protein expression was observed in pET-22b(+)-GhWRKY91 and pET-32a(+)-GhWRKY91 in BL21(DE3) and pMAL-c5x-GhWRKY91 in Arctic-Express™(DE3)RP. However, the protein expressed by pGEX-4T-1-GhWRKY91 in Arctic-Express™(DE3)RP was presented in the supernatant, and a soluble protein could be obtained. Therefore, GhWRKY91 soluble protein can be successfully obtained by constructing GhWRKY91 gene into the prokaryotic expression vector of pGEX-4T-1.
Key words:  cotton  GhWRKY91gene  SDS-PAGE  prokaryotic expression  expression analysis

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