莱茵衣藻CrPGP3和CrFAT1基因克隆及其序列分析与原核表达

Cloning,sequence analysis,and prokaryotic expression of Chlamydomonas reinhardtii gene CrPGP3 and CrFAT1

克隆莱茵衣藻(Chlamydomonas reinhardtii)的CrPGP3和CrFAT1基因,并对其序列进行生物信息学分析和原核表达。选取莱茵衣藻CC124提取总RNA后,反转录c DNA,PCR获得CrPGP3和CrFAT1的全长编码区,构建原核表达载体CrPGP3-PGEX-6p-1和CrFAT1-PGEX-6p-1,转入大肠杆菌DL21(DE3)中,在37℃、1.0mmol/L IPTG诱导整合蛋白表达。结果表明,CrPGP3和CrFAT1的全长编码区分别为786 bp和1 188 bp,分别编码261和395个氨基酸;CrPGP3是与脂类合成相关磷脂酰甘油磷酸合成酶(Phosphatidylglycerophosphate synthase)属于CDP-alcohol phosphatidyltransferase家族,CrFAT1是酰基载体蛋白硫脂酶(Acyl-ACP thioesterase)属于hot dog家族;SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致。本文成功克隆了CrPGP3和CrFAT1基因的全长编码区,并在原核生物中表达得到了预期蛋白。

In order to express the CrPGP3 and CrFAT1 genes of Chlamydomonas reinhardtii in prokaryotic cell, we cloned and analyzed these two sequences. After the total RNA in C. reinhardtii CC124 was extracted, the revers transcription were performed. The full length cDNA obtained by PCR was inserted into the prokaryotic expression vector PGEX-6p-1 and transformed into Escherichia coli BL21 （DE3） . These positive clones were called CrPGP3-PGEX-6p-1 and CrFAT1-PGEX-6p-1. The target proteins were expressed after adding 1.0 mmol/L IPTG. The results showed that the CrPGP3 and CrFAT1 gene had 786 bp and 1 188 bp coding 261 and 395 amino acids, respectively. Sequence analysis indicated that CrPGP3 was a member of super family ＂CDP-alcohol phosphatidyltransferase＂ , and the CrFAT1 belonged to a member of ＂hot dog＂ super family. The SDS-PAGE displayed that the expressed proteins were consistent with the anticipated size. The recombinant plasmids expressed the target proteins in E. coli BL21.