Cloning of self-incompatibility SFB gene from‘Xiaobaixing'apricot(Prunus armeniaca L.)and its plant expression vector construction

【Objective】To lay a foundation for self-incompatibility improvement and self-compatibilityapricot cultivars breeding by plant gene engineering technology.【Methods】The SFB(S-haplotype-specif-ic F-box protein)gene was cloned by RT-PCR(Reverse Transcription PCR)combined with 3′RACE(Rapid Amplification of c DNA Ends)from pollen sample which came from the self-incompatible apricot‘Xiaobaixing'(Prunus armeniaca L.‘Xiaobaixing')in Xinjiang province. Plasmids p MD19-T Vectorwere adopted for cloning of PCR products,p BS-T restriction enzyme cutting site ofthis Vector were used.To construct the sense expression vector of SFB gene p CAMBIA-S-SFB,we combined the ORF of SFB positively with expression vector p CAMBIA-35S-MCS-NOS-NPTII between Kpn I and Sac I site by re-striction enzyme ligation;to construct the antisense expression vector p CAMBIA- A- SFB,we recombined the ORF(Open Reading Frame)of SFB reversely with plant expression vector between Kpn I and Sal I site;the recombinant expression vector plasmids were transferred into Agrobacterium tumefaciens LBA4404 by eletroporation.【Results】We obtained a 1 136 bp 3′-end full c DNA sequence(Submitted toGen Bank and under review)which contained a 912 bp complete ORF,the deduced amino acid sequencecontained a F-box domain,two variable region(V1,V2),and two hypervariable region(HVa,HVb),which had similar structural characteristics with SFB in the other Rosaceae plants. The restriction enzymedigestion results showed that the ORF of SFB was correctly inserted into p CAMBIA-35S-MCS-NOSNPTII sites between 35 S promoter and terminator,the two plant expression vectors p CAMBIA-S-SFB and p CAMBIA-A-SFB were constructed and transferred into Agrobacterium tumefaciens LBA4404 suc-cessfully by specific primer PCR identification.【Conclusion】These results provide expression vectors forself-incompatibility improvement and genetic transformation of self-compatibility apricot cultivars breed-ing by gene engineering technology.