Construction Of Paeonia Somatic Embryogenesis Related Gene PsSERK Overexpression Vector And Genetic Transformation

Somatic embryogenesis receptor-1ike kinase(SERK)gene in the embryonic callus of herbs such as carrot,rice and woody plants such as cocoa are specific expression,which is formed in the process of somatic embryo early occurred.SERK gene is considered to be one of molecular markers of plants of embryonic callus early occurred.On the basis of Ps SERK which has been cloned from the peony,the study used the callus induced by the plantlets' s petiole of Tree peony cultivars Paeonia ostii ‘Fengdan' as test material to build the overexpression vector of Ps SERK.Then respectively transformed it into the model plant Arabidopsis thaliana and peony callus by agrobacterium mediated genetic transformation system.And preliminary identified the transgenic plant,which Laid the foundation for analysis and identification of Ps SERK gene function through the chip technology and expression analysis technique.The main results were as follows: 1.PCR amplification of the full-length sequence of Ps SERKUsing specific primers and high-fidelity DNA polymerase to clon Ps SERK gene full-length sequence by RT-PCR amplification from peony callus.Sequencing relevant plasmids of Ps SERK c DNA clon,the results showed that the c DNA sequence similarity was 98.67% compared to acquired Ps SERK and Genebank.The coding amino acids sequence similarity was 98.72% compared to acquired Ps SERK and Genebank.2.Construction of Ps SERK overexpression vector and transformation of agrobacteriumAccording to construction scheme of expression vector,using two restriction enzymes Hind? and Eco R? to cut the DNA plasmid of PBI221 and p CAMBIA1301.Fragment of 35S-GUS-NOS and p CAMBIA1301 linear plasmid vector were obtained respectively.Then connecting the purpose fragment to the linear plasmid vector to form recombinant plasmid vector,and transferring the recombinant plasmid vector into DH5 ? cells.After screening and validation,intermediate cloning vector was built,which was named p CAMBIA1301-35S-NOS.Using Xba? and Sac? to cut p CAMBIA1301-35S-NOS,Spe? and Sac? to cut DNA plasmid of acquired Ps SERK.Connecting the products to form recombinant plasmid and transforming the recombinant plasmid vector into DH5? cells.After screening and validation,Ps SERK overexpression vector was built.The overexpression vector was transferred into agrobacterium LBA4404 and GV3101 by freeze-thaw methods.3.Genetic transformation of Arabidopsis thaliana by agrobacteriumPs SERK overexpression vector was transferred into Arabidopsis thaliana by floral dip method.The transgenic seedlings which have the resistance of Hyg were acquired by preliminary screening.The correctness of Ps SERK overexpression vector was verified by phenotypic characterization and PCR detection.Agrobacterium LBA4404 and GV3101 were filtered to found either LBA4404 or GV3101 was better strains of arabidopsis genetic transformation.The hygromycin sensitivity of Arabidopsis thaliana was tested to inspect different concentrations of Hyg tolerance.The results showed that concentration of Hyg 25mg·l-1 is fit for Arabidopsis thaliana.4.Genetic transformation of peony callus by agrobacteriumAccording to the preliminarily established genetic transformation system of peony callus(Infection by agrobacterium LBA4404,the agrobacterium thallus was suspended by plants broth.We used the Bacilli OD600=0.6 to infect directly in 20 min,put the infected callus in co-culture medium in dark 3 days at 22?.Then rinsed and soaked the callus in 30 min by 200mg·l-1 Cef.After that,put it in liquid proliferation medium with 100mg·l-1 Cef,meantime shaked 24 h.Then removed the callus,washed it and put it in the solid selective medium with 100mg·l-1 Cef and 5mg·l-1 Hyg once in 20 days.),Ps SERK overexpression vector was transfered into the peony callus.The peony callus pieces were in blue by GUS's tissue staining method.And GUS's transient expression activity was detected successfully.The results showed that peony callus had GUS activity sites,which preliminary showed that Ps SERK overexpression vector has been transferred into the peony callus.