小麦麦谷蛋白高分子量亚基N端序列克隆及原核表达分析  

Cloning and prokaryotic expression analysis of N-terminal sequence of high molecular weight subunit of wheat gluten

作　　者：王志成 王琦 张长付[1] 张聪聪 王泽濠 张霞 王金水[1] WANG Zhicheng;WANG Qi;ZHANG Changfu;ZHANG Congcong;WANG Zehao;ZHANG Xia;WANG Jinshui(College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China)

机构地区：[1]河南工业大学生物工程学院,河南郑州450001

出　　处：《河南工业大学学报（自然科学版）》2023年第4期60-66,共7页Journal of Henan University of Technology：Natural Science Edition

基　　金：国家粮食和物资储备局粮油食品工程技术研究中心开放课题(GA2017001);河南工业大学高层次人才科研启动基金项目(2018BS044);河南工业大学校基金项目(2018QNJH21)。

摘　　要：为了探究麦谷蛋白高分子量亚基(HMW-GS)的N-末端结构域对面粉品质的影响,以HMW-GS的1Dx5的N-末端结构域(1Dx5-N)为目的基因进行序列克隆及原核表达,研究大肠杆菌BL21(DE 3)为载体菌进行转化,鉴定阳性克隆,以IPTG和乳糖作为诱导剂,并通过正交试验,筛选最佳的诱导条件。结果表明:1Dx5-N序列基因全长267 bp,共编码氨基酸89个,预测分子质量为16.211 kDa;经转化、阳性克隆鉴定后,证明了该表达载体能够在大肠杆菌中稳定遗传;IPTG和乳糖均可诱导1Dx5-N进行表达;SDS-PAGE表明1Dx5-N重组蛋白多为包涵体蛋白;IPTG诱导蛋白最佳表达条件为温度30℃,浓度0.6 mmol/L,诱导时间16 h;乳糖诱导蛋白最佳表达条件为温度30℃、质量浓度9 g/L、诱导时间16 h。通过对比发现,乳糖可代替IPTG诱导1Dx5-N蛋白表达。To investigate the effect of the N-terminal structural domain of high molecular weight wheat glutenin subunit(HMW-GS)on flour quality,the N-terminal structural domain of 1Dx5 of HMW-GS(1Dx5-N)was used as the target gene for sequence cloning and prokaryotic expression.Escherichia coli BL21(DE 3)was used as carrier bacteria for transformation,positive clones were identified,IPTG and lactose were used as inducers,and the optimal induction conditions were screened by orthogonal test.The results showed that the total length of the 1 Dx5-N sequence gene was 267 bp,encoding 89 amino acids,and the predicted molecular weight was 16.211 kDa.After transformation and positive cloning,it was proved that the expression vector could be stably inherited in Escherichia coli.Both IPTG and lactose could induce the expression of 1Dx5-N.SDS-PAGE analysis showed that the 1Dx5-N recombinant proteins were mainly inclusion body proteins.The optimal expression conditions of IPTG-induced protein were 30℃,0.6 mmol/L,and 16 h.The optimal expression conditions of lactose induced protein were 30℃,9 g/L and 16 h.It was found that lactose could induce the expression of 1Dx5-N protein instead of IPTG.

关 键 词：麦谷蛋白高分子量亚基 1Dx5-N 末端结构域 原核表达 

分 类 号：TS201.2[轻工技术与工程—食品科学]