| Dendrobium officinale is an important herb of orchid family in China.Its main medicinal ingredients are indole alkaloids,polysaccharides and amino acids.MVA pathway is one of the important pathways for the generation of indoles alkaloid precursors of terpenoids.3-hydroxyl-3-methyl glutaryl coenzyme A reductase(HMGR)and mevalerate kinase(MK)are the key rate-limiting enzymes in the cytoplasmic MVA pathway,and their studies are very important.In this paper,full-length cDNA and promoter sequences of DoHMGR and DoMK were cloned,and MYB family members were screened to enrich the database of MYB transcription factors,so as to lay a theoretical foundation for further studies on terpenoid synthesis of Dendrobium officinale.The main results are as follows:1.Using the method of homologous cloning and RACE to clone DoHMGR and DoMK(GeneBank login number:MF536713).Tissue expression and induced expression analysis showed that DoHMGR and DoMK were expressed in various tissue parts of Dendrobium officinale,and responded to salicylic acid(SA)and abscisic acid(ABA)induced expression in the induced expression of protocorm.2.DoHMGR and DoMK promoter sequences were cloned,and software analysis showed that the promoter region contained a variety of cis-acting elements,including several MYB binding elements.3.101 R2R3-MYB genes were screened and divided into 25 subfamilies(S1-S25)according to cluster analysis,and bioinformatics analysis was performed.Six subfamily members were selected for tissue expression analysis and hormone induced expression analysis.Homologous cloning was performed on two of them to obtain DoMYB29 and DoMYB31 gene sequences.4.The eukaryotic expression vectors of pCAMBIA 1301-DoHMGR and pCAMBIA 1301-DoMK were successfully constructed.Agrobacterium-mediated transformation of wild arabidopsis thaliana resulted in the screening of stable overexpressed T3-generation plants. |
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