The Cloning,Transformation Of Chalcone Isomerase And The Cloning Of MYB Regulator In Erigeron Breviscapus

Erigeron breviscapus(Vant.)Hand.-Mazz.is an very important herb of Erigeron of compositae, which contains flavonoids as its main activity ingredient. As an very importan kind of econdary metabolites, the flavonoids demonstrate special physiological activity and multiple biological functions in plants. Therefor, the clone of structural genes concerning in flavanoids and its controlling elements, and study its role in biosynthesis pathway, became the hot topic in botany, genetics,pharmacology,plany physiology and plant nutriology, et al. So far, many research in the world, had revealed that the structural gene and its concerning controlling element, especially the MYB transcription factors,not only in structure, but also in function, have an very conservative tendency during evolution. Therefor, the information about its structural genes and regulator factors of a plant which had not studed incompletely, can be obtained easily by the methods through bioinformatics analysis based on data of public database of model plants. Basing on the information from bioinformatics, one can study the strcture and function of gene and regualting factor involved in flavonoids biosynthesis pathway of plant, which was absence about its genetical backgroud, via through gene cloning, gene transformation and the data of phenotype variation of transgenic plant.Chalcone isomerase(CHI) is key gene in flavonoids biosynthesis pathway. And the R2R3-MYB transcription factors which have putative regulation during flavonoids synthesis. In our study, we gained sequence information of CHI and MYB screening from the transcriptome data via bioinfromatics analysis. And the full-length c DNA are obtained via RACE cloning. After a comprehensive analysis of biological information, we constructed a recombinational plasmid by combining PBI121-EGFP with CHI or MYB gene thus to acquire the fusion-expression of CHI or MYB with GFP protein. Then transferred the recombinational plasmid PBI121-EGFP-CHI into the leaves of Erigeron breviscapus by Agrobacterium-mediated transformation so that EGFP-CHI fusion-expression gene can integrated with the genome of Erigeron breviscapus. During this process, we alsocan build up a system of genetic transformation in Erigeron breviscapus. This research may be conducive to a profound comprehension of gene’s structure and function involved in flavonoid biosynthesis and pathway. The obtained results of this study are included as follows:1.Cloning and bioinformatics analysis of CHI and MYBIn our study, we gained CHI and MYB full-length gene via RACE amplification method. CHI and MYB have ORF as 717 bp and 780 bp, they respectively encode238 and 260 amino acids. After homology and evolutionary analysis, CHI gene closely related to the activity of chalcone isomerase involved in flavonoid biosynthesis. The conservative domain of MYB is[W]-x(20)-[W]-x(20)[W]-x(32)[W]-x(19)-[W], which belongs to R2R3-MYB family,it’s function also associated with flavonoid biosynthesis.2.Construction and transformation of CHI and MYB recombinantConstructed a recombinational plasmid by combining PBI121-EGFP with CHI or MYB gene and transferred to Agrobacterium GV3101 via electroporation method.3. Establishment of genetic transformation system in Erigeron breviscapusTo ensure an appropriate concentrations of selective agentia and bacteriostatic agentia which are not only inclined to attain the experimental purpose effectively but also affect the seedling at a minimum, we disposed leaves of Erigeron breviscapus at different gradients of kanamycin(Kan) and cephalosporin(Cef), according to the results, we chose Kan 5 mg/L and Cef 200 mg/L to be the most appropriate selective and bacteriostatic agentia. We then transferred the recombinational plasmid PBI121-EGFP-CHI into the leaves of Erigeron breviscapus via Agrobacterium GV3101 and observed crown galls after selective cultivating.