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目的分离红花天冬氨酸代谢途径关键酶天冬氨酸激酶(AK)基因的全长cDNA序列,进行植物超表达载体构建。方法根据红花种了转录组文库注释和qRT-PCR鉴定的AK基因核心片段及表达分析数据,采用RACE技术获得红花AK(CtAK)基因的全长cDNA,利用DNA重组技术构建植物超表达载体。结果生物信息学分析表明,CtAK基因全长1 703bp,ORF区为1 626 bp,编码541个氨基酸残基,功能结构域分析推测,该基因可能为单功能反馈抑制敏感植物AK1。通过DNA重组技术成功构建以35 S为启动子和Bar抗性基因并含有叶绿体转运肽的pCAMBIA3301-CTP-AK1植物超表达载体。结论获得CtAK1基因的编码序列并构建植物超表达载体,为进一步研究其生物学功能及其在红花氨基酸代谢调控中的作用机制奠定基础。
OBJECTIVE To isolate the full length cDNA of aspartokinase (AK), a key enzyme involved in the aspartate metabolism pathway in Crocus sativus, and to construct a plant overexpression vector. Methods The full-length cDNA of safflower AK (CtAK) gene was obtained by RACE and the plant overexpression vector was constructed by DNA recombination technology based on the cDNA library of safflower seed and the core fragment of AK gene identified by qRT-PCR. . Results Bioinformatics analysis showed that the CtAK gene was 1 703 bp in length and 1 626 bp in ORF region, encoding 541 amino acid residues. The functional domain analysis suggested that this gene may be a single functional feedback suppression sensitive plant AK1. The pCAMBIA3301-CTP-AK1 plant overexpression vector with 35S promoter and Bar resistance gene and chloroplast transit peptide was successfully constructed by DNA recombination technology. Conclusion The coding sequence of CtAK1 gene and construction of plant overexpression vector were obtained, which laid the foundation for further study of its biological function and its mechanism in the regulation of amino acid metabolism in safflower.