油菜GDSL脂肪酶基因BnLIP1的克隆及其原核表达

Cloning of a GDSL Lipase Gene from Brassica napus and Preliminary Study of its Recombinant Expression in Escherichia coli

克隆油菜GDSL脂肪酶基因BnLIP1,实现该基因在原核系统中重组表达,为研究其功能奠定基础。采用TRIzol法提取总RNA,通过RT-PCR法扩增BnLIP1编码序列,构建原核表达载体pEL1并转化大肠杆菌,IPTG诱导表达后通过SDS-PAGE检测目的蛋白。根据GenBank报道的序列设计引物,从萌发的油菜种子黄化幼苗中成功克隆到BnLIP1的编码序列,长度为1170bp。将该编码序列构建到原核表达载体pET32a(+)并与标签序列融合,在E.coliBL21菌株中成功表达了与标签蛋白融合的BnLIP1蛋白,大小约为61kDa。首次实现了油菜GDSL脂肪酶基因在原核系统中的表达,为GDSL脂肪酶Bn-LIP1蛋白的分离纯化及活性研究奠定了基础。

To isolate coding sequence of a GDSL-motif lipase gene from Brassica napus L. designated as BnLIP1, over-express it in prokaryotic systems, the following methods were used, TRIzol-method for total RNA isolation, one step RT-PCR, construction of recombinant expression vector, recombinant expression induced by isopropyl-D-thiogalactopyranoside （IPTG）, and SDS-PAGE. Primers specific to BnLIP1 were de- signed, and its coding sequence of 1170bp was successfully amplified with total RNA as template from etiolated seedlings. The expression construct pET32a （＋） carrying the BnLIP1 coding sequence was preformed, and transformed into E. coli BL21 used as a host. Induced by IPTG, BnLIP1-Tag fusion protein with molecular weight of 61 kDa was successfully expressed in transformant. It is the first time to demonstrate recombinant expression of rape GDSL-motif lipase gene in prokaryotic system, which paves the way for uncovering its biochemical activity in the future.