Cloning, Expression And Construction Of Expressive Vectors Of Genes Involved In The Anthocyanin Biosynthesis In Lycoris Radiata

The Lycoris species are good kinds of bulbiferous ornamental plants, which are widely distributedin China, and are widely applied in the cut flowers, groundcover greening and configuration oflandscape. In this research, the crucial genes involved in the anthocyanin biosynthesis were cloned fromLycoris radiata, and the expression pattern of those genes were analysed and plant expressive vectors ofthree genes were constructed, which results layed the foundation for the future research on molecularmechanism of color formation and cultivating new varieties of flower using genetic engineeringbreeding method of Lycoris radiate. The main results of the research are as follows:Five complete-length cDNAs of LrCHI, LrFLS, LrF3H, LrDFR and LrANS were isolated byhomologous cloning and RACE technology from Lycoris radiate. The cDNA sequences were1360bp,1293bp,1338bp and1292bp and included a whole open reading frame encoding236,333,365,364and355amino acids respectively. Sequence comparative analysis using NCBI blastn and blastp toolsshowed that those five genes shared a high homology with relevant genes of other plants. The molecularweight, theoretical pI, signal peptide, transmembrane domain, second structure and conserved structuraldomain of those five genes were analysed by bioinformatics software. The phylogenetic tree showedthat the system evolution of those genes were consistent with plant taxonomy and had obvious featuresof genus and species.The expression of LrCHI, LrFLS, LrF3H, LrDFR and LrANS genes in different developing stagesof flower and different organs were carried on analysis by Quantitative RT-PCR. The results showedthat LrCHI and LrDFR had a high expression level in the early stages of flower however the expressionlevel of LrF3H and LrANS were raised rapidly and then decreased with the developing of flower. Theexpression level of LrFLS was higher in the wither period than any other period. The results ofexpression analysis in different organs showed that LrCHI and LrFLS had the similarity expressionfeature that the expression level in leaves, scapes and petals all higher than roots and corms. In themeanwhile, the expression level were high in roots, corms and leaves but lower in petals and scapes.In order to made further research about the function of LrF3H, LrDFR and LrANS theoverexpression vectors were constructed. LrF3H, LrDFR and LrANS were connected to the plantexpressive vector pCAMBIA13011which had a CaMV35S promoter, and then constructedpCAMBIA13011-LrF3H、pCAMBIA13011-LrDFR and pCAMBIA13011-LrANS successfully. On thebasis of that results, the engineering strain of Agrobacterium tumefaciens EHA105were constructedsuccessfully.