Gene Cloning,Vectors Construction And Analysisof Interacted Proteins Of Flavonoids Biosynthesis Genes

Rose(Rosa rugosa) with beautiful, fragrant, colorful flowers, is an ornamental shrub of rosaceous. it has high application value in landscaping and industrial production because of its unique flavor, flower color and resistance. Petunia(Petunia hybrida) is a model plant in plants gene engineering. As an important secondary metabolites in plants, flavonoids is concerned with flower color, stress resistance and auxin transport. For human, flavonoids has anti-inflammatory, antioxidative and other physiological functions. In recent years, it has also been a focus of research to explore the flavonoids metabolic pathway for improving traits of plants and human health.CPC transcription factor amonged to MYB transcription factors in Arabidopsis thaliana, it regulated expression of structural genes to affect flavonoids biosynthesis. In order to explore the complex regulation network of the flavonoids metabolic pathway, proteins were identified which were interacted with CPC protein from Arabidopsis thaliana transcription factors library by Yeast Two-Hybrid; we had a preliminary research on flavonoids biosynthesis genes through gene cloning, vectors construction. The results were as follows:1. One protein which may be interacted with CPC protein was identified from Arabidopsis thaliana transcription factors library by Yeast Two-Hybrid. At CPC gene was cloned using At CPC-p CAMBIA2300 s plasmid as a template, PGBKT7-At CPC bait vector was constructed and the vector was transformed into yeast Y187. At GATA28 gene was identified from Arabidopsis thaliana transcription factors library by Yeast Two-Hybrid, the protein encoded by At GATA28 gene may be interacted with CPC protein.2. Six flavonoids biosynthesis genes were cloned from rose and petunia, and overexpression vectors were constructed in our study. Based on transcriptome sequencing results about Rosa multiflora, Rr C4 H, Rr CHS, Rr F3 H and Rr CHI genes were cloned using rose c DNA as a template by homology cloning strategy, p MOG22-Rr C4 H, p MOG22-Rr CHS, p MOG22-Rr F3 H and p CAMBIA2300s-Rr CHI overexpression vectors were constructed; Ph PAL and Ph4 CL gene sequences were obtained from NCBI, Ph PAL and Ph4 CL genes were cloned using petunia c DNA as a template, p CAMBIA2300s-Ph PAL and p MOG22-Ph4 CL overexpression vectors were constructed, the overexpression vectors were transformed into agrobacterium EHA105.3. Four tobacco RNAi vectors were constructed in our study. Based on transcriptome sequencing results about Nicotiana tabacum, Nt LAR, Nt ANS, Nt ANR and Nt FLS gene sequence were obtained, Nt FLS and Nt(LAR+ANR) RNAi vectors were constructed by Gateway; Nt LAR and Nt ANS ami RNA vectors were constructed. The RNAi vectors were transformed into agrobacterium EHA105.