The Cloning And Functional Analysis Of CYP81E8s Gene In Hosta

As an important traditional medicine,studies on Hosta are limited to the aspects of physiology and pharmacology.Its flower color and fragrance are important traits of landscaping,while flower color and fragrance are complex traits controlled by multiple genes.However,the flower color of Hosta is only white and purple,and only some varieties have strong sweet fragrance,so it is particularly important to study the influencing factors in its gene regulation process.In this study,H.’So Sweet’ was used as experimental material.Through data analysis of transcriptome database,the expression pattern of obtained HsCYP81E8 s gene sequence was analyzed,and the similarities and differences between the two genes were compared by bioinformatics analysis.In order to explore the function of HsCYP81E8 s in flower color and floral fragrance,VIGS transient silencing vector and transient overexpression vector were constructed.The flower buds of stage of Hosta were infected with Agrobacterium-mediated vacuum injection and the effects of gene silencing and overexpression on flower color and floral fragrance were analyzed by quantitative fluorescence PCR.In order to avoid the defect of transient transformation of genes,Arabidopsis thaliana was used for allogenic transformation to obtain stable transgenic Arabidopsis thaliana genetic lines,and then the influence of overexpression of HsCYP81E8 s gene on flower color and floral fragrance was explored.The specific results of the study are as follows:1.The expression pattern analysis of HsCYP81E8 s showed that the expression level of HsCYP81E8 s gene was the highest in the blooming stage of H.’So Sweet’.2.The HsCYP81E8 and HsCYP81E8-1 from the transcriptome database of H.’So Sweet’ was successfully cloned,which were 1527 bp and 1071 bp respectively,encoding 508 and 356 amino acids,respectively.By comparison,only the 5’ end of the two proteins was different,and both of them were typical CYP450 family proteins.3.VIGS and transient overexpression techniques showed that whether genes were silenced or expressed,the expression of key enzyme genes in anthocyanin synthesis reduced,affecting anthocyanin formation;The expression level of TPS,the key enzyme gene for terpenoids,showed a synergic effect.When the expression level of HsCYP81E8 and HsCYP81E8-1 genes increased,the expression level of TPS genes also increased to affect the synthesis of Hosta flower fragrance.4.Arabidopsis thaliana was used as a dip-flower heterologous transformation model to obtain stable transgenic high-expression strains of Arabidopsis thaliana.Phenotypic analysis showed that the growth of both transgenic plants was more vigorous than that of the wild type,indicating that genes may be involved in regulating plant growth and development.Fluorescence quantitative PCR showed that overexpression of both genes decreased the expression of ANS gene in flavonoid metabolic pathway and TPS gene in terpene metabolic pathway,indicating that overexpression of these genes inhibited the synthesis of flower color and floral fragrance in Arabidopsis thaliana.