Cloning And Functional Analysis Of Flavonoids Related Genes Of Saussurea Involucrate Kar.et Kir

Object: Saussurea involucrate Kar. et Kir. have complex composition of secondary metabolism, which pharmacological activity focus on flavonoids, alkaloids snd polysaccharides, in which flavonoids are the main active ingredient. Flavonoids of Saussurea involucrate Kar. et Kir. was used by several minorities in China, because it have some special Pharmaceutical effects. Belongs to the plant secondary metabolites.However, due to the slow pace of its natural growth, combined with people of their excessive excavation,on the verge of depletion of existing wildlife resources, biological diversity under serious threat. Therefore,look for wild Saussurea involucrate Kar. et Kir alternatives to meet the market demand has become an urgent issue.Methods: we cloned the sik P and Sik TTG1 gene by RT-PCR and transformed it into Saussurea involucrate Kar. et Kir. and tomatoes by Agrobacteria tumefaciens-mediated transformation. Transgenic lines were confirmed by PCR, and the expression of the transgene was confirmed by RT-PCR, By measuring the content of flavonoids and analysis transgenic plants and controlled plant’s flavonoids content.Results:(1)Sequence analysis showed that the c DNA of Sik P contained a 783 bp ORF encoding a polypeptide of261 amio acids, Sik P gene and Saussurea Medusa Maxim Sm P gene sequence homology of 96.5 %,encoding a polypeptide of 261 amio acids, Amino acid homology is above 96.9 %. amio acids with a calculated molecular mass of 29 086.5 Da and a theoretical p I of 7.64, asthe basic amino acid, no span membrane structure, was a hydrophilic protein. Instability parameter of 61.23, belongs to the unstable protein. Phylogenetic analysis further showed that Saussurea involucrate Kar. et Kir. Sik P had nearest relationship with Saussurea Medusa Maxim Sm P is in the same branch.(2)Sequence analysis showed that the c DNA of Sik P contained a 1011 bp ORF encoding a polypeptide of337 amio acids, Sik TTG1 gene and Saussurea Medusa Maxim Sm TTG1 gene sequence homology of97.14 %, Amino acid homology is above 95.55 %. The protein encoded by with WD40 domain structure,and belongs to the members of the family of WD40, amio acids with a calculated molecular mass of 37692.3 Da and a theoretical p I of 4.82, asthe acidic amino acid, no span membrane structure, was a hydrophilic protein. Instability parameter of 52.10, belongs to the unstable protein. Phylogenetic analysis further showed that Saussurea involucrate Kar. et Kir. Sik TTG1 had nearest relationship with Saussurea Medusa Maxim Sm TTG1 is in the same branch.(3) Obtained by PCR of sik P and sik TTG1 gene, constructed the plant expression vector p BI121-Sik P and p CAMBIA2300-Sik TTG1, and then integrated them into Saussurea involucrate Kar. et Kir. and tomatoes genome by Agrobacterium-mediated, screened using Kan, identified by PCR and RT-PCR, and finally obtained transgenic plants.(4) Measured transgenic Saussurea involucrate Kar. et Kir.’s flavonoids content and tomato leaf’s flavonoids content, compared with the controls, the results found that Sik P gene of transgenic Saussurea involucrate Kar. et Kir. flavonoids content is 9.85 times that of the control group, the Sik P gene transgenic tomato leaf flavonoids content is 5.4 that of the control group times, the Sik TTG1 gene transgenic tomato leaf flavonoids content is 3.95 that of the control group times.Conclusion: To sum up, the Saussurea involucrata Kar. et Kir. Myb transcription factors Sik P gene and WD40 transcription factors Sik TTG1 gene plays an important of plant secondary metabolism roleregulation,And it can effectively improve the content of flavonoids.