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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-length cDNA of dihydrodipicolinate synthase (DHDPS), a key enzyme in the lysine biosynthesis pathway, and construct a plant overexpression vector. Methods The sequence of CtDHDPS gene was obtained from the cDNA library of safflower transcriptome. The full length c DNA sequence of CtDHDPS gene was cloned from safflower seeds by RT-PCR and RACE. The p BASTA-CtDHDPS plant overexpression vector was constructed by DNA recombination technology. Results The full length of CtDHDPS gene was 1 309 bp with a 954 bp open reading frame encoding 317 amino acids. The calculated theoretical isoelectric point was 5.93 and the relative molecular mass was about 34 750.79. The plant overexpression vector p BASTA-CtDHDPS was successfully constructed by DNA recombination technology. Conclusion The full length c DNA sequence of CtDHDPS gene was obtained and the plant overexpression vector was successfully constructed. This study may provide a scientific basis for elucidating the biological function of CtDHDPS gene and its mechanism in the process of lysine biosynthesis.