红花转录因子CtMYB1基因的克隆及原核表达  

Cloning and prokaryotic expression of transcription factor Ct MYB1 gene in Carthamus tinctorius

作　　者：官丽莉[1,2] 张雪[1,2] 韩怡来 朱栋[2] 杨晶[1,2] 杜林娜[1,2] 王法微[1,2] 李海燕[1,2] 李校堃[1] 

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

出　　处：《中草药》2015年第17期2603-2609,共7页Chinese Traditional and Herbal Drugs

基　　金：国家高技术研究发展计划("863")项目(2011AA100606);国家自然科学基金资助项目(31201237;31101172);吉林省科技厅中青年领军人才及优秀创新团队项目(20111815);教育部博士点基金-青年教师基金项目(20122223120002);大学生创新创业项目(201410193045)

摘　　要：目的从红花Carthamus tinctorius花瓣中克隆转录因子基因Ct MYB1,并进行序列分析和原核表达载体的构建。方法根据红花转录组测序结果中得到的高表达的Unigene123933序列,利用RT-PCR和RACE技术从红花花瓣中扩增得到Ct MYB1基因的全长c DNA序列,并进行生物信息学分析;以Ct MYB1基因的全长c DNA序列为模板,PCR扩增得到Ct MYB1基因的开放阅读框(ORF)序列,构建原核表达载体p EASY-E1-Ct MYB1。转化大肠杆菌BL21(DE3)进行诱导表达。结果成功从红花中克隆1个MYB基因,命名为Ct MYB1,Gen Bank登录号为KJ524853。Ct MYB1基因全长893 bp,ORF为750 bp,编码249个氨基酸。同时,成功构建了该基因的原核表达载体。SDS-PAGE结果显示该蛋白的大小约30 000,与预测的蛋白相对分子质量一致。结论成功克隆了Ct MYB1基因,构建了原核表达载体,初步证明该基因在大肠杆菌中成功表达。Objective To obtain a transcription factor gene MYB, clone a Ct MYB1 gene from the safflower petals of Carthamus tinctorius, and performe its sequence analysis and prokaryotic expression vector construction. Methods According to high expression Unigene123993 sequence in safflower transcriptome, MYB gene was cloned from safflower by RT-PCR and RACE methods. The full-length c DNA sequences Ct MYB1 gene as templates, the open reading frame（ORF） of c DNA sequences was obtained by PCR. Prokaryotic expression vector p EASY-E1-Ct MYB1 was constructed and the expression in E. coli BL21（DE3） was transformed. Results A MYB gene was successfully cloned from the safflower petals of C. tinctorius, named Ct MYB1（Gen Bank accession No. KJ524853）. A full length c DNA of Ct MYB1 was 893 bp and ORF was 750 bp, encoding a protein of 249 amino acid. The prokaryotic expression vector was obtained. SDS-PAGE results showed that the molecular weight was 30 000, same with the relative molecular weight of predicted protein. Conclusion Ct MYB1 is cloned from safflower and the prokaryotic expression vector is constructed, which preliminarily proves that the gene is successfully expressed in E. coli.

关 键 词：红花 MYB CtMYB1 原核表达 红花黄色素 RT-PCR RACE 

分 类 号：R282.12[医药卫生—中药学]