目的 从红花Carthamus tinctorius 花瓣中克隆转录因子基因CtMYB1,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CtMYB1基因的全长cDNA序列,并进行生物信息学分析;以CtMYB1基因的全长cDNA序列为模板,PCR扩增得到CtMYB1基因的开放阅读框(ORF)序列,构建原核表达载体pEASY-E1-CtMYB1。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CtMYB1,GenBank登录号为KJ524853。CtMYB1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CtMYB1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 CtMYB1 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 cDNA sequences CtMYB1 gene as templates, the open reading frame (ORF) of cDNA sequences was obtained by PCR. Prokaryotic expression vector pEASY-E1-CtMYB1 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 CtMYB1 (GenBank accession No. KJ524853). A full length cDNA of CtMYB1 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 CtMYB1 is cloned from safflower and the prokaryotic expression vector is constructed, which preliminarily proves that the gene is successfully expressed in E. coli.

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)