红花DHDPS基因全长cDNA的克隆及植物表达载体构建

Cloning of full-length cDNA of gene encoding dihydrodipicolinate synthase from Carthamus tinctorius and construction of plant expression vector

目的克隆红花赖氨酸生物合成途径关键酶二氢吡啶二羧酸合酶(dihydrodipicolinate synthase DHDPS)基因的全长c DNA序列,构建植物超表达载体。方法根据红花转录组文库注释信息获得红花DHDPS(CtDHDPS)基因的中间序列,通过RT-PCR与RACE技术从红花种子中克隆CtDHDPS基因全长c DNA序列。采用DNA重组技术构建p BASTA-CtDHDPS植物超表达载体。结果分离CtDHDPS基因全长1 309 bp,开放阅读框954 bp,编码317个氨基酸,编码蛋白理论等电点为5.93,相对分子质量约为34 750.79。通过DNA重组技术成功构建了植物超表达载体p BASTA-CtDHDPS。结论获得CtDHDPS基因全长c DNA序列并成功构建植物超表达载体,为阐明CtDHDPS基因的生物学功能及其在红花赖氨酸生物合成途径中作用机制提供科学依据。

Objective To clone the full-lengthcDNA sequence of DHDPS gene encoding the key enzyme DHDPS in lysing biosynthesis pathway of Carthamus tinctorius, and to construct the plant expression vector. Methods The dihydrodipicolinate synthase（DHDPS） gene fragment was acquired according to the sequence of transcriptome in C. tinctorius, and the full-lengthcDNA sequence of CtDHDPS gene from C. tinctorius seeds was cloned by RT-PCR and RACE technologies. The p Basta-DHDPS plant expression vector was constructed using traditional molecular cloning and recombination technique. Results Bioinformatics analysis showed that the full-lengthcDNA of CtDHDPS was 1309 bp, open reading frame was 954 bp, encoding a polypeptide of 317 amino acids, the theoretical isoelectric point of the coded protein was 5.93, and the molecular weight was about 34 750.79. The plant expression vector p Basta-DHDPS was successfully constructed by traditional molecular cloning and recombinant technique. Conclusion The full-length sequence of CtDHDPS gene is obtained and the plant expression vector is successfully constructed, which lays a foundation for the further study on the mechanism of CtDHDPS in regulation of essential amino acid metabolism.