Study On The Function Of PbPAT10 Gene Of Pear And The Acquisition Of Transgenic Dwarf Plants

Pear dwarf dense planting has gradually become the development trend of pear tree cultivation at home and abroad.Choosing suitable dwarf rootstock is one of the important factors affecting the realization of the pear dwarf dense planting cultivation mode.Pyrus betulifolia is the most widely used root stock in pear producing areas in north China.It has good characteristics such as strong adaptability,strong resistance to stress,and strong affinity for grafting with eastern and western pears,but it does not have dwarf effect.Therefore,the breeding of turmeric rootstock with dwarf effect has become an urgent problem to be solved in the development of pear cultivation in China.With the development of gene editing technology,it is possible to improve a trait of rootstock directionally.Protein palmitacyltransferase 10(PAT)is a type of acylation in post-translational modification of proteins,which significantly influences the growth and development of plants.Previous studies have shown that the loss of pat10 gene function leads to dwarf traits in plants.In this experiment,Pyrus betulifolia and ’new pear no.7’ were used as test materials.The leaf regeneration system of ’new pear no.7’was initially established,and then the genetic transformation system of ’new pear no.7’ leaf was constructed.The PbPAT10 genes of Pyrus betulifolia and ’new pear no.7’ were cloned and sequenced,and the function of PbPAT10 genes of Pyrus betulifolia was verified in yeast and arabidopsis thaliana.At the same time,CRISPR/Cas9 expression vectors corresponding to PbPAT10 gene of Pyrus betulifolia and ’new pear no.7’ were constructed,and resistant plants of Pyrus betulifolia and ’new pear no.Twere obtained,in order to clarify the function of pear PAT10 gene and to obtain pear mutant plants with dwarf traits for the next step,so as to provide a new way for genetic improvement of dwarf pear dense planting.The main research results are as follows:1.It was verified that PbPAT10 gene of Pyrus betulifolia and arabidopsis thaliana have the same function-dwarf functionThe stable expression vector of PbPAT10 gene was constructed and recovered in AtPAT10 heterozygous mutant(T-dna insertion)of arabidopsis thaliana.The results showed that the arabidopsis strain height of transformed Pyrus betulifolia PbPAT10 was significantly higher than that of pure and mutants,and was close to that of arabidopsis WT Col-0.A point mutation was performed on cysteine 194th in the PAT10 enzyme active region of duolie.Through yeast complementary test,it was found that PbPAT10 gene with pear point mutation could not restore the activity of yeast,while normal PbPAT10 gene could restore the activity of yeast.It was clear that PbPAT10 gene of pear point mutation was similar to AtPAT10 gene of arabidopsis thaliana.2.The genetic transformation system of ’new pear no.7’ was preliminarily establishedThe in vitro regeneration system of ’new pear no.7’ was established.By adjusting the concentration and ratio of 6-BA,IBA,TDZ and NAA,the plant medium formula suitable for the regeneration of ’new pear no.7’ was screened out:NN69 22.2g/L+6-BA 1.0mg/L+TDZ 1.0mg/L+NAA 0.2mg/L+AGAR 8g/L.The genetic transformation system of ’new pear no.7’ was constructed.In the transformation system,the leaves of ’new pear no.7’ were co-cultured and culturate d sucsively.The culture medium was NN69 22.2g/L+6-BA 1.0mg/L+TDZ 1.Omg/L+N AA 0.2mg/L+AGAR8g/L,and the dark culture medium was NN6922.2g/L+6-BA1.0mg/L+TDZ 1.Omg/L+NAA 0.2mg/L+AGAR 8g/L.The culture medium was NN69 22.2g/L+6-BA1.0mg/L+TDZ 1.0mg/L+NAA 0.2mg/L+AGAR 8g/L+Kan 20 mg/L+Cef 400 mg/L,and dark culture was conducted until the emergence of seedlings.3.The pear PAT10 gene CRISPR/Cas9 expression vector was constructed and pear resistant plants were obtainedThe PbPAT10-2 CRISPR/Cas9 expression vectors of Pyrus betulifolia and’New pear No.7’ were successfully constructed.The experiment designed two targets.The sgRNA expression cassettes of Pyrus betulifolia PbPAT10-2 and ’New pear No.7’ PbPAT10-2 genes were constructed by Overlapping PCR method,and then the sgRNA expression cassette and pCRISPR/Cas9P35S-N plasmid were subjected to restriction enzymes Bas I and Under the joint action of T4 ligase,two CRISPR/Cas9 expression vectors of PbPAT10-2 with kanamycin selection as marker genes were obtained.Five resistant seedlings of Pyrus betulifolia Pear and four resistant seedlings of ’new pear no.7’were obtained by using the regeneration of du pear leaves and the regeneration of ’new pear no.7’leaves.4.The preparation of Pyrus betulifolia protoplasts determined that the optimal enzymolysis time was 12h,and the combined effect of cellulase and pectinase was better than that of segregation enzyme and cellulase.