Cloning and sequence analysis of the SFBB-γ gene in 'Kuerlexiangli' pear

Abstract：【Objective】'Kuerlexiangli'pear, which belongs to Rosaceae Pomoideae, is a unique and famous cultivar in the Xinjiang Uyghur Autonomous Region.Most species in Rosaceae exhibit S-RNasebased gametophytic self-incompatibility (GSI) .The pollen-part determinant, pollen S, has long remained elusive.Pollen S in Prunus is an F-box protein gene (SLF/SFB) located near S-RNase.Identification of the S locus F-box brothers (SFBB) in Pyrus and Malus suggested that the multiple F-box genes were pollen S candidates because they exhibited pollen specific expression, S haplotype-specific polymorphisms, and linkage to the S locus.In Pyrus, three SFBBs were identified from a single S haplotype.The SFBBs were homologous to other haplotype genes of the same group (i.e., -α, -β and-γ groups) .The objective of this experiment was to clone the full-length sequence of the SFBB-γ gene, which was involved in self-incompatibility in'Kuerlexiangli'pear.This work provides a foundation for future studies on the mechanism of self-incompatibility and the functional identification of SFBB genes in Pomoideae.【Methods】The pollen was collected from'Kuerlexiangli'pear trees grown at the Luntai National Fruit Germplasm Resource Garden in the Xinjiang Region.The'Kuerlexiangli'pear SFBB gene sequence (GQ456943) was referenced in NCBI, and six pairs of gene-specific primers and eight RACE nested primers were designed.The primers were used with RT-PCR and RACE (rapid amplification of cDNA ends) to clone one pollen-specific, full-length SFBB-γ gene.DNAman software was used to predict and analyze the amino acid sequence.The biological information was analyzed using ProtParam and other online prediction software.The complete sequences of SFBB genes in all Rosaceae Pomoideae and the complete sequences of self-incompatibility SFB genes in some Rosaceae Prunus were selected from NCBI.Multiple alignment was done using Clustal X software and different gene names of the same sequence were excluded.The most recently published name of the gene was used and a phylogenetic tree was constructed using MEGA 4.0 software.【Results】Two pairs of gene specific primers[B3 F (5'-TCATCCTCCACTTGTATC-3') /B3 R (5'-TAAATCCGTCATCGTAG-3') and B6 F (5'-ATGTCTCAGGTGCGTGAAAGTG-3') /B6 R (5'-TAAATCCGTCATCGTAG-3') ] and two nested primers of 3'RACE [Outer3 (5'-AACTATTGTACCGTTTCTAAAGGATGG-3') /Inner5 (5'-ATGGGTAATGGACTACGATG-3') ] were selected from the six pairs of gene specific primers and eight RACE nested primers.One full-length SFBB-γ gene (1 227 bp) was successfully cloned using RT-PCR and RACE technology.The linkage between the SFBB-γ gene and the S-RNase gene of'Kuerlexiangli'pear has not yet been confirmed.Therefore, the gene was named SFBBx-γ (registered with GenBank) .The DNAman software predicted that SFBBx-γ contained one complete open reading frame of 1 191 bp, encoding 396 amino acids.ProtParam and other software predicted that the protein was unstable, hydrophilic, and non-secreted.The protein was predicted to be mainly involved in binding energy metabolism and transport and it can function as either ligase or lyase.Amino acid cluster analysis showed that69 pollen-specific SFBB and SFB genes related to self-incompatibility in Rosaceae were divided into two categories.In the group of SFBB genes, all of the Pyrus SFBB-γ genes were clustered into a single subclass.Within this subclass, SFBBx-γ was most closely related to the SFBB-4 gene, which has also been identified in'Kuerlexiangli'pear.The SFBB-α genes and the SFBB-β genes were clustered into one subclass, which included four SFBB genes and three SFLB genes in Malus.The group of SFB genes included 11 SFB genes in Prunus and the only known SFB-v gene in Pyrus.【Conclusion】One fulllength, pollen-specific SFBB-γ cDNA gene was cloned from'Kuerlexiangli'pear.This c DNA gene included an F-box region and four variable regions (V1, V2, V3, and V4) .Its length was 1 127 bp and it contained one complete 1 191 bp ORF which encoded 396 amino acids.Phylogenetic tree analysis showed that the SFBB genes of Malus and the SFBB genes of Pyrus were clustered into one group.In this group, the SFBB-α and SFBB-β genes of Pyrus were clustered into one sub-class.The SFBB-γgenes were clustered into another sub-class.The features of the SFBB genes suggest that they are good pollen S candidates.However, it is not clear if all of the multiple SFBBs in a haplotype are involved in pollen S specificity.The possibility that none of the SFBBs are pollen determinant can not be excluded at present.However, SFBB-γ genes of Pyrus share high levels of amino acid identity among haplotypes.Low-or no-allelic polymorphism does not exclude the SFBB genes from being the component of pollen S if the SI system is of the'non-self recognition by multiple factors' type.Self-incompatibility in Prunus is considered to represent a'self recognition by a single factor'system.In contrast, Japanese pear (Pyrus) has a'non-self recognition by multiple factors' SI system.In Japanese pears, multiple SFBB genes may have a synergistic effect on the mechanism of self-incompatibility.Therefore, it is still not possible to exclude the possibility that SFBB-γ gene is a pollen S gene in Pyrus.Additional research needs to be done to verify the function of these genes.