番茄内质网ω-3脂肪酸去饱和酶基因原核表达载体的构建与鉴定

Construction and Identification of an Endoplasmic Reticulum-localized Tomato Omega-3 Fatty Acid Desaturase(LeFAD3) Gene Prokaryotic Expression Vector

通过RT-PCR的方法从番茄叶片克隆到内质网ω-3脂肪酸去饱和酶(LeFAD3)基因的部分编码区,该片段cDNA为309 bp,将其克隆到pET-30a(+)载体中,酶切位点分别是BamHⅠ和SacⅠ,构建了原核表达载体pET-LeFAD3,并在大肠杆菌BL21中表达融合蛋白,经IPTG诱导蛋白表达,提取蛋白并采用SDS-PAGE和蛋白质免疫印迹法检测目的蛋白的表达情况。酶切鉴定结果表明,LeFAD3基因原核表达载体构建成功,目的蛋白成功表达。

The coding region length of LeFAD3 gene about 309 bp nucleotides was isolated by RT-PCR from tomato leaves and was subcloned into the pET-30a（＋） vector between the BamHⅠ and SacⅠsites.A recombinant of prokaryotic expression vector pET-LeFAD3 was constructed and transformed into E.coli BL21 and then expressed by inducing with IPTG,transducted into BL21 and the exogenous protein expression was induced by IPTG.The exogenous LeFAD3 expression was examined by SDS-PAGE and Western immunoblotting.Results showed that LeFAD3 gene was successfully constructed,expression of LeFAD3 gene in E.coli could provides the basis for further research on LeFAD3 function.