| Transcription factors participate physiological and biochemical processes by activating or inhibiting the genes’ expression in plants. There are many transcription factor families in plants, where MYB gene family is one of them. It had been discovered that MYB proteins play an vital role in multiple growth phase in plant development. For example, MYB transcriptiors regulate plants secondary metabolism, affects the plant cell shape and mediates plants response mechanism to stress. However, previous research on MYB transcription factors has only focused on the analysis of their physiological function and the function of single MYB gene, little has been done on the gene control network. Thus the research related to the complicated regulation network of MYB genes is of great importance.To explore this regulation gene network Yeast One-Hybrid system and Yeast Two-Hybrid system were usually used. In the experiment, MYB7, one member of MYB gene family, was selected as the research subject, A Yeast One-Hybrid AD-cDNA library based on Rosa chinensis’Slater’s crimson China’ as the experiment materials were constructed. The promotor of MYB71.86kb upstream sequence of MYB7gene were isolated by FPNI-PCR. The detailed experiment contents and results are as followed:1. We use the buds of Rosa chinensis’Slater’s crimson China’ as experimental materials, constructed a Yeast One-Hybrid cDNA library. We evaluated this cDNA library via transformation efficiency and the length of recombination fragments. The transformation efficiency is1.4х106,the recombination fragments is800bp-1000bp。2. We use the MYB7overexpression vector PMY-RrMYB7plasmid as a template, cloning the full length of cDNA of MYB7gene, constructed the bait vector for Yeast Two-Hybrid and transformed the vector to Y187.3In order to explore the role of MYB7gene in transcriptional activation pathway, the promoter of MYB7gene was isolated by new genome walking FPNI-PCR.In the end,1.86kb fragment after three reaction was cloned. |
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