红花2-甲基-6-叶绿基-1,4-苯醌甲基转移酶基因的克隆及表达分析

目的克隆红花维生素E合成相关关键酶2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)基因,并进行生物信息学及表达分析,为红花维生素E生物合成及调控机制研究奠定基础。方法根据红花种子转录组数据库中得到的中间序列,采用RT-PCR和RACE方法从红花种子中克隆MPBQ MT基因序列,通过生物信息学对该基因蛋白的特征进行分析,构建MPBQ MT与相关物种MPBQ MT的系统进化树,利用RT-PCR方法分析在红花种子不同发育时期MPBQ MT基因的表达量。结果MPBQ MT基因全长1 392 bp,命名为Ct MPBQ MT,具有完整的开放阅读框(ORF),共1 038 bp,编码345个氨基酸。生物信息学分析显示,该基因编码的蛋白理论相对分子质量约为38 900。保守结构域预测表明,该基因编码的蛋白具有典型的SAM蛋白功能结构域。结合其他物种的MPBQ MT基因构建系统树表明,红花MPBQ MT基因与其他物种氨基酸具有一定的同源性,其中与向日葵和生菜同源性高达89%和86%。实时荧光定量PCR分析表明,MPBQ MT基因在红花开花后50 d的种子中表达量最高。结论成功地对MPBQ MT基因进行克隆及表达分析,为红花维生素E合成及调控机制研究奠定基础。

Cloning and expression analysis of MPBQ MT gene in safflower

Objective Safflower (Carthamus tinctorius) is an important natural source of vitamin E. 2-Methy-6-phytyl-1,4- benzoquinone methyltransferase (MPBQ MT) is a key enzyme in vitamin E synthesis pathway. MPBQ MT gene was cloned, bioinformatics was analyzed, and expression was analyzed to provide the foundation for the biosysthesis and regulation mechanism of vitamin E in safflower. Methods According to the intermediate sequence obtained from the database of the safflower seed,MPBQ MT gene sequence was cloned by RT-PCR and RACE techniques, and the protein characteristics were analyzed using bioinformatics and constructing phylogenetic tree. The expression of MPBQ MT gene in the different development stages was analyzed using real time-PCR. Results The full cDNA sequence of MPBQ MT gene was 1 392 bp, named CtMPBQ MT, contained an open reading frame (ORF, 1 038 bp), and encoded a protein of 345 amino acids with a predicted molecular mass of 38 900. The conserved structural domain analysis showed that it had the typical functional domains of SAM protein. Sequence alignment and phylogenetic tree analyses showed that the MT MPBQ gene had some homology with other amino acids, and among them CtMPBQ MT had 89% and 86% of consistency with MPBQ MT of Helianthus annuus and Lactuca sativa. The expression of CtMPBQ MT gene in safflower seeds at different development stages was determined by quantitative real-time PCR, it was found that the highest expression level of CtMPBQ MT gene was detected in 50 d after flowering. Conclusion MPBQ MT gene of safflower is successfully cloned, analyzed, and expressed, meantime a basis for the study on matter in the synthesis and regulation of vitamin E is provided.