Cloning and expression analysis of MPBQ MT gene in safflower / 中草药

ABSTRACT

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.