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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。
Objective To clone the MPBQ MT gene, a key enzyme involved in the synthesis of safflower vitamin E, and analyze its bioinformatics and expression profile. E biosynthesis and regulatory mechanisms laid the foundation for the study. Methods The sequence of MPBQ MT gene was cloned from safflower seeds by RT-PCR and RACE according to the middle sequence obtained from the safflower seed transcriptome database. The characteristics of MPBQ MT gene were analyzed by bioinformatics to construct MPBQ MT The phylogenetic tree of species MPBQ MT was used to analyze the expression of MPBQ MT gene at different developmental stages of safflower seed using RT-PCR. Results The MTB gene of MPBQ was 1 392 bp in length and named Ct MPBQ MT. It contained a complete open reading frame (ORF) of 1 038 bp encoding 345 amino acids. Bioinformatics analysis showed that the theoretical molecular weight of the protein encoded by this gene was about 38 900. Conserved domain prediction shows that the protein encoded by this gene has a typical SAM protein functional domain. The phylogenetic tree based on the MPBQ MT gene of other species showed that MPBQ MT gene had some homology with amino acids of other species, of which the homology with sunflower and lettuce was as high as 89% and 86%. Real-time PCR analysis showed that MPBQ MT gene was the highest in the seeds at 50 d after flowering. Conclusion The successful cloning and expression analysis of MPBQ MT gene laid the foundation for the study on the synthesis and regulation of safflower vitamin E.