红花单功能反馈抑制敏感型CtAK1基因克隆、序列分析及表达载体构建  

Molecular cloning,sequence analysis,and construction of expression vector of monofunction feedback inhibition sensitive CtAK1 gene in Carthamus tinctorius

作　　者：王艳芳[1,2,3] 张玲[1,3] 张娜[2] 张娟[2] 李红群[2] 杨晶[1,2] 杜林娜[2] 官丽莉[1,2] 李海燕[1,2] 李校堃[1,2,3] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]吉林农业大学生命科学学院,吉林长春130118 [3]吉林农业大学中药材学院,吉林长春130118

出　　处：《中草药》2015年第24期3740-3745,共6页Chinese Traditional and Herbal Drugs

基　　金：国家高技术研究"863"发展计划(2011AA100606);国家自然科学基金资助项目(31401445);吉林省科技厅医药产业推进计划项目(20140311034YY);教育部博士点基金(20122223120002)

摘　　要：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 separate the full length cDNA of gene encoding the key enzyme AK in aspartate metabolic pathways of Carthamus tinctorius and to construct plant overexpression vector. Methods According to annotation on transcriptome library of C. tinctorius and core fragments and expression analysis data of CtAK identified by qRT-PCR, we separated the full length cDNA of AK gene of C. tinctorius （CtAK） using RACE technology and constructed plant expression vector using recombinant DNA technology. Results Bioinformatics analysis showed the full length CtAK was 1 703 bp and ORF area was 1 626 bp, encoding 541 amino acid residues. The function structure domain analysis showed the gene might be a monofunction feedback inhibition sensitive AK1 from plant. We successfully constructed plant expression vector pCAMBIA3301-CTP-AK1 which contained 35 S promoter and Bar resistance genes and chloroplast transit peptide by recombinant DNA technology. Conclusion The gene encoding CtAK1 is obtained and the plant overexpression vector is constructed, which lays the foundation for researching on biological function and mechanism of action in amino acid metabolism regulation of C. tinctorius.

关 键 词：红花 天冬氨酸激酶 RACE 生物信息学 植物表达载体 

分 类 号：R282.12[医药卫生—中药学]