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作 者:黄卓[1] 王丕武[1] 张卓[1] 马建[1] 代力强[1]
出 处:《吉林农业大学学报》2013年第5期547-551,共5页Journal of Jilin Agricultural University
基 金:吉林省科技发展计划项目(215-00011)
摘 要:为转BADH基因玉米品系Western杂交等蛋白水平检测做前期准备,对辽宁碱蓬中的甜菜碱脱氢酶基因(BADH)进行原核表达、纯化及功能性鉴定。通过构建含有BADH基因的原核表达载体转化至大肠杆菌BL21中,诱导其进行原核表达,并对表达产物进行Ni-NTA柱亲和层析纯化分析。所构建pET28a-BADH原核表达载体经PCR和双酶切鉴定及测序分析与预期结果一致;SDS-PAGE结果显示表达产物约50.5 kD,且为可溶性;生物功能性鉴定BADH蛋白的表达使受体细胞在高盐条件下的抗性有显著提高。This experiment was conducted to make early preparations for the detection of BADH trans- genic maize lines Western blot. Prokaryotic expression, purification and functional identification of be- taine aldehyde dehydrogenase (BADH) gene were performed. Prokaryotic expressed vectors of BADH gene were constructed and transformed into Escherichia coli B[21, and BADH gene was induced to ex- press by IPTG compound. The expressed product was purified by Ni - NTA affinity column chromatogra- phy and the salt-tolerance of the prokaryotic expressed vectors was identified. The result of identification of prokaryotie expressed vector PET28a - BADH by PCR reaction and double enzyme digestion and se- quence analysis was consistent with the expectation. The result of SDS - PAGE shows that the target pro- tein was about 50.5 kD and soluable. The expression of BADH protein by biological functional identifica- tion obviously improved salttolerance of transformed cell.
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