摘要
丝氨酸乙酰转移酶(serine acetyltransferase,SAT)是甲硫氨酸和半胱氨酸合成途径的关键酶,利用该基因提高作物中甲硫氨酸水平具有广阔的应用前景。为了研究拟南芥SAT1(Arabidopsis thaliana SAT1)基因的蛋白表达情况及相关功能,构建了带有His标签的AtSAT1基因原核表达载体p ET-30b(+)-SAT1-His(6)。将其导入大肠杆菌BL21(DE3)pLysS中并用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,经固定化金属离子亲和层析(IMAC)纯化获得了抗原蛋白SAT1-His(6)。该抗原蛋白经免疫兔子,成功制备了具有较高特异性和灵敏性的SAT1蛋白多克隆抗体。Western blotting表明,该抗体可用来检测转AtSAT1基因的转基因玉米株系中SAT1蛋白的积累量,为后期对该基因开展进一步深入研究具有重要意义。
As a key enzyme in methionine and cysteine biosynthetic pathway, serine acetyltransferase(SAT) has huge potential to increase methionine content in crops. To investigate the Arabidopsis thaliana SAT1(AtSAT1)protein accumulation and function in maize transgenic lines, the His-tagged prokaryotic expression vector pET-30b(+)-SAT1-His(6)of AtSAT1 was made and transformed into Escherichia coli expression strain BL21(DE3)pLys S. After IPTG(isopropyl-β-D-thiogalactoside) induction, the recombinant SAT1 His-tagged protein was purified by immobilized metal ion affinity chromatography(IMAC). Rabbit polyclonal antibodies were produced and analyzed using Western blot. The analysis showed that the prepared antibodieswere of high specificity and sensitivity and therefore can be used to detect AtSAT1 accumulation in transgenic maize lines, thus providing a reference for the further study of SAT1 gene function.
出处
《分子植物育种》
CAS
CSCD
北大核心
2016年第6期1369-1375,共7页
Molecular Plant Breeding
基金
四川省财政基因工程青年基金项目(2015GXSC-004)
农业部转基因专项重点项目(2014ZX0800301B)共同资助
关键词
拟南芥
丝氨酸乙酰转移酶
原核表达
多克隆抗体
Arabidopsis thaliana
Serine acetyltransferase
Prokaryotic expression
Polyclonal antibody
