Construction Of Paeonia PsSERK Interference Expression Vector And Its Function Preliminary Research

Paeonia suffruticosaisa traditional flowerof our country,it's famous for viewing and medicinal plants.In the study of peony tissue culture,we found that it was difficult totransformnon-embryogenic callus into embryogenic callus in the process of somatic embryogenesis,and there was coexistencephenomenon of embryogenic callus with non-embryogenic callus.Therefore,increasing the conversion rate of non-embryogenic callus changing to embryogenic callushas become an urgent problem needed to be solved in the establishment of somatic embryo regeneration system of peony.At present,it is concluded that somatic embryogenesis receptor-like kinase(SERK)gene plays an important role in the induction of plantcallus,and itcan increase the incidence of embryogenic callus ofplantssignificantly.SERK gene can be used as one of the molecular markers of embryogenic callusoccurred early.The study was based on the preliminary work,using the callus tissue induced by the petiole of the peony cultivar 'Fengdanbai' in vitro as the materials.At first,optimized the peony callus genetic transformation system,constructed the Ps SERK interference expressionvector by the system of CRISPR/Cas9.The Ps SERK overexpression vector and interference expression vector were transferredinto the peony callus respectively by agrobacterium mediated genetic transformation system,and determined the related hormone,enzyme activity index,and used the real-time fluorescent quantitative PCR technology to analyse the expression of Ps SERK in different carriers.The main results and conclusions are as follows: 1.Optimization of agrobacterium-mediated genetic transformation system of peonyIn the transgenic experiment on callus of Peony,experimented the antibacterial effect with different concentrations of bacteriostatic agent(Cef,Cb),and counted the growth status.The results showed that,the effect of antibacterial was Cb to agrobacterium tumefaciens LBA4404 in the agrobacterium tumefaciens-mediated transformation system of Peony callus,and the suitable concentration is 200mg·L-1.2.Construction of the Ps SERKinterference expression vectorAccording to the experimental protocol,according to the full-length c DNA sequence of Ps SERK gene,design targetsequence.The target was cloned into the psg R-Cas9-At vector backbone through the Bbs I site,and form the intermediate cloning vector Ps SERK-psg R-Cas9-At.Analysis the restriction enzyme cutting site of Ps SERK-psg R-Cas9-At and p CAMBIA1300,hrough Hind IIIand Kpn Idouble enzyme digestion,and connect,then form a recombinant plasmid.Successfully constructedthe Ps SERK interference expression vector pCAMBIA1300-Ps SERK-psg R-Cas9-At.And the constructed interfering expression vector was transferredinto Agrobacterium tumefaciens LBA4404 by freeze-thawing method.3.Agrobacterium tumefaciens-mediated transformation of Paeonia callusThe Ps SERK overexpression vector and the interference expression vector were transferred respectively into callus of peony,used the method of optimized agrobacterium tumefaciens callus genetic transformation.GUS gene histochemical staining method was used to experiment,the results showed that,detected successfullytransient expression activityof GUS,and the callus fragments were blue.Preliminary showed thatthe Ps SERK overexpression vector was transferred into Peony callus.The c DNA of transgenic callus was used as template to PCR amplification,and obtained the bands was the same as expected,while the negative control showed no band,which indicated that the Ps SERK interference expression vector was successfully transferred into the Peony callus.4.Preliminary analysis of Ps SERK gene functionAt first,the Ps SERK overexpression and interference expression vector were respectively transferred into the peony callus,and exogenous add cytokinin 6-BA(0.5 mg·L-1),TDZ(0.1 mg·L-1)was no transgenic callus(control)as the research object.Respectively collect callus of above three treatments at 0,5,10,15,20,25,30,35,40 and 45 days.Determined the related hormone(ABA,GA3,IAA),enzyme activity(POD,SOD,APX,CAT)index,and real-time fluorescence quantitative analysis.The results showed that,the expression of Ps SERK gene in the callus of the three treatments,the order from high to low was: overexpression> no transgenic(control)> interference,and reached the highest value at 30 d.Ps SERK gene plays an important role in the enzymes metabolism and hormone expression in the process of somatic embryogenesis of Peony.It is speculated that embryogenic cells may begin to appear at 20-25 d in culture,and at 25-35 d may have a large number of embryogenic cells.Then the Ps SERK interference expression vector was transferred into the model plant arabidopsis thaliana.The results showed that,the interference expression of Ps SERK gene can cause arabidopsis thaliana slow growth and delayed flowering characteristics,which has great influence on the growth and flowering time of Arabidopsis thaliana.