Cloning And Genetic Transformation Of LcCHSs And LcDFRs Genes From Loropetalum Chinense Var.rubrum

Loropetalum chinense var.Rubrum is a perennial plant belonging to the genus Loropetalum in the Hamameliaceae.Its leaves will remain red for a long time due to sufficient light.Lack of light makes the leaves appear green.Because of its strong adaptability,easy reproduction,drought tolerance,pruning tolerance,etc.,it has become a commonly used garden ornamental plant in southern my country.As an important genetic trait of ornamental plants,flower color has a very important influence on the ornamental value and economic value of plants.The different colors of plants are closely related to the anthocyanins formed in plants.A variety of synthases are involved in the biosynthetic pathway of plant anthocyanins,among which chalcone synthase is the first restriction enzyme for the synthesis of flavonoids,which is produced by catalyzing the substrates coumayl and malonyl.The reaction further synthesizes the intermediate chalcone.Dihydroflavonol-4-reductase is a key enzyme in the flavonoid metabolic pathway leading to the synthesis of anthocyanins.It plays an important role in catalyzing the reduction of substrate flavanones to leuco anthocyanins.In order to study the molecular mechanism of the anthocyanin biosynthesis of Loropetalum chinense,two LcCHSs gene sequences were cloned and analyzed by bioinformatics.This paper uses RT-PCR technology to clone the LcCHSl and LcCHS2 genes of Loropetalum chinense.The ORF of LcCHS1 is 1170 bp,encoding 389 amino acids,and the ORF of LcCHS2 is 1182 bp,encoding 393 amino acids.The amino acid sequence of the two LcCHSs is more than 83%identical to the amino acid sequence of CHSs derived from tea tree,grape,tobacco,etc.And the three residues(Cys164,His303 and Asn336)involved in CHS enzyme catalysis and two important CHS enzyme sequences("RLMMYQQGCFAGGTVLR" and "GVLFGFGPGL")in the amino acid sequence of the two LcCHSs are strictly conserved,indicating that CHS is evolving Conservatism on the function.The tertiary structure prediction shows that both LcCHSs can form homodimers,and the spatial structures of the two LcCHSs proteins are very similar.At the same time,LcCHSI-PS1300 and LcCHS2-PS 1300 expression vectors were constructed.The two LcDFRs gene sequences of Loropetalum chinense were cloned,their bioinformatics information was analyzed,and the tobacco leaf transient expression method was used to observe the subcellular localization of the protein.The ORF of LcDFR1 is 1014 bp,encoding 337 amino acids,and the ORF of LcDFR2 is 993 bp,encoding 330 amino acids.The amino acid sequence identity of DFRs between LcDFRs and grape,peony,and peony is between 68%-84%,indicating that LcDFRs have high sequence conservation.Phylogenetic tree analysis showed that LcDFRl clustered with the DFRs of peony and peony,while LcDFR2 clustered with the DFRs of tobacco and tomato.Subcellular localization analysis showed that LcDFR1 and LcDFR2 were mainly cytoplasmic proteins.The expression vectors LcDFR1-PS1300 and LcDFR2-PS1300 were also constructed.In this thesis,through the cloning and bioinformatics analysis of the LcCHSs and LcDFRs genes of Loropetalum chinense,and preliminary analysis of the functions of LcCHSs and LcDFRs genes,it provides certain theoretical information for the molecular mechanism of the flower color formation of Loropetalum chinense,and then provide a theoretical basis for enriching the flower color of Loropetalum chinense and improving the ornamental value.