Cloning Of Self-incompatibility S-locus Gene In Korla Fragrant Pear

Object: Korla fragrant pear(Pyrus sinkiangensis Yü)is a unique and famous cultivar in the Xinjiang Uyghur Autonomous Region.Self-incompatibility which exhibits in Korla fragrant pear has restricted the development of its industrialization.The objective of this experiment was to clone the full-length sequence of self incompatibility genes related to S-locus in Korla fragrant pear by RT-PCR and RACE.This work provides a foundation for mechanism of self-incompatibility research and self-compatibility pear cultivars breeding by analysising phylogenetic tree of S gene in Pyrus.Methods: 1?Cloning of S-RNase allelic genes in Korla fragrant pear.The primers of S gene's conservative sequence of Pear were referenced to design gene-specific primers.Pistils were collected from Korla fragrant pear.The full length sequences of S-RNase allelic genes were obtained by RT-PCR and RACE.The characteristics of putative proteins were analyzed using Protparam software.The complete sequences of S-RNase genes in all Rosaceae Pyrus were selected from NCBI and a phylogenetic tree was constructed by MEGA 4.0.2 ? Cloning of the SFBB gene in Korla fragrant pear and 9 different pear cultivars.The Korla fragrant pear SFBB gene sequence(GQ456943)was referenced in NCBI,and six pairs of gene-specific primers and eight RACE nested primers were designed.One pair of gene specific primer(B3F/B3R)and two nested primers(Outer3/Inner5)of 3'RACE were selected from those primers.The full length sequence of SFBB gene was obtained from pollon of Korla fragrant pear by RT-PCR and RACE.The full-length sequence primer of SFBB gene in Korla fragrant pear was used to clone DNA sequences of SFBB genes in 9 different pear cultivars.The characteristics of putative proteins were analyzed using Protparam software.The complete sequences of SFBB genes in all Rosaceae Pomoideae were selected from NCBI and a phylogenetic tree was constructed by MEGA4.0.Results: 1 ? Two full-length DNA and c DNA of S22-RNase gene(Gen Bank Acceptance Number:KX214125)and S28-RNase gene(Gen Bank Acceptance Number: KX214124)were cloned from Korla fragrant pear.The full-length DNA of S22-RNase was 1052 bp and the full length c DNA was904 bp which contained an ORF(open reading frame)of 684 bp and encoded a protein of 227 amino acids.The full length DNA of S28-RNase was 1148 bp and the full length c DNA was 921 bp which contained an ORF of 687 bp and encoded a protein of 228 amino acids.It is predicted that both of the two genes had the RNase-T2 motif by BLASTP.The phylogenetic analysis indicated that S22 and S28 from Korla fragrant pear laied different branch and possessed high polymorphism.Amino acid cluster analysis showed that intraspecific homology of S-RNase genes in Pyrus was not higher than interspecific homology,which indicated that the S-RNase gene was formed before the formation of the species.2?One full-length SFBB gene(1344 bp)was successfully cloned from Korla fragrant pear.This gene contained one complete ORF of 1191 bp,encoding 396 amino acids and was named SFBBx-gamma(Gen Bank Acceptance Number: KU756268).Eight full-length DNA sequences of SFBB genes were successfully cloned from 9 different pear cultivars.Six of them were new genes,named SFBBx1-gamma-SFBBx6-gamma(registered with Gen Bank),respectively.Prot Param and other software predicted that the proteins were unstable,hydrophilic,and non-secreted.The proteins were predicted to be mainly involved in binding energy metabolism and transport and it can function as either ligase or lyase.The complete sequences of SFBB genes in all Rosaceae Pomoideae were selected from NCBI.Amino acid cluster analysis showed that 65 pollen-specific SFBB genes related to self-incompatibility in Rosaceae maintain a high level of similarity at the structural level.The similarity of SLFB/SFBB genes in apple and SFBB gene in pear was not affected by the difference of the species,which indicated that the SFBB gene was formed before the formation of the species.Conclusion: The full-length sequences of S22/28-RNase and SFBBx-gamma were successfully cloned from Korla fragrant pear,and six full-length DNA sequences of new SFBB-gamma genes were cloned from 9 different pear cultivars.This work provides a foundation for mechanism of deeply self-incompatibility research and S gene information of self-compatibility pear perfection by analysising two phylogenetic trees of S-RNase genes in Pyrus and SFBB genes in Pomoideae.