The Cloning And Functional Verification Of HVCHS And HVANS Genes In Hosta Ventricosa

Hosta is a kind of perennial herbaceous flower that belongs to hosta genus of Liliaceae.It is widely used as an ornamental and medicinal plant.In the past,Hosta was mostly planted in Jiangnan region of China,and then widely introduced and cultivated.Now it has developed into an important flower with coappreciation of flowers and leaves in the north of country.Although there are many varieties of hosta,but the color is vary single,with only white and purple.Therefore,it is important to excavate the genes that control the metabolism of the flower color of hosta,and then to cultivate the new color of hosta.The main pigments of flowe color are betalains,carotenoids and flavonoids,among them flavonoids are the most widely available pigments in plants.Chalcone synthase and Anthocyanin synthase are the key enzyme in flavonoid biosynthesis pathway.A large number of studies have shown that chalcone synthase and Anthocyanin synthase play important roles in the formation of plant flower color.In this study,the cDNAs clone encoding CHS and ANS were cloned by RT-PCR techniques from petals of Hosta Ventricosa,and named HVCHS and HVANS,the HVCHS and HVANS cDNAS were 1176bp and 1059bp long and encoded 391 and 352 amino acids respectively.The structure and function of HVCHS and HVANS genes were predicted by bioinformatics.Plant overexpression vectors pC AMBIA3 301-HV CHS and pC AMBI A3 301-HVAN S were transformed into tobacco by agrobacterium-mediated method.The function of HVCHS and HVANS genes were verified by measuring molecular detection and physical and biochemical indications of transgenic tobacco plants to lay a foundation for the research on hosta flower The detailed achievements of this rsearch are showd as below:(1)CHS and ANS genes were extracted from the hosta transcriptome database and specific primers were designed according to ORF.The coding region sequences of HVCHS and HVANS genes were amplified by RT-PCR.After sequencing comparison,the homology was 100%.(2)Through ligation of HVCHS and HVANS genes with linearized pCAMBIA3301 vector,the plant over-expression vectors pCAMBIA3301-HVCHS and pCAMBIA3301-HVANS were successfully constructed,then,after sequencing comparison,the homology was 100%.(3)Total RNA,was extracted from roots,stems,lesves,flowers and the flowers of different developmental stages(green bud,purple green interbud,purple bud,early flowering,full flowering).The expression of HVCHS and HVANS genes were analyzed by real-time fluorescence quantitative method.The results showed that the HVCHS and HVANS genes were highly expressed in flowers and increased with the deepening of flower color,and had a positive correlation with anthocyanin content.(4)The over-expression vectors pCAMBIA3301-HVCHS and pCAMBIA3301-HVANS were transformed into tobacco by using agrobacterium-mediated method.Through herbicide screeing and PCR detection,transgenic tobacco with HVCHS gene and transgenic tobacco with HVANS gene were obtained.The transcription and expression of HVCHS and HVANS genes in tobacco genome were detected by fluorescence quantitative PCR.The results showed that the flower color of transgenic tobacco was deepened and the content of anthocyanin and flavonoids were increased.