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Molecular Cloning and Characterization of PbSFBB13-gamma from Chinese White Pear (Pyrus bretschneideri)

中国白梨PbSFBB13-gamma基因的分子克隆与序列特征*


以5个已知S基因型的中国白梨品种为试材,设计分别对应于梨SFBB-alpha, SFBB-betaSFBB-gamma基因的3对引物对这5个品种进行基因组PCR扩增。结果仅引物组合PSFBG-F/PSFBG-R从‘金花’(S13S18),‘金花四号’(S13S18)和‘鹅梨’(S13S34)中扩增出一约1 300 bp的条带,经回收、克隆、测序后,鉴定其为SFBB-gamma基因,命名为PbSFBB13-gammaPyrus bretschneideri SFBB13-gamma),GenBank登录号为EU081892。逆转录PCR结果表明PbSFBB13-gamma仅在花粉中特异表达; 序列分析表明该基因不含内含子,编码区含1 191个核苷酸,编码396个氨基酸,预测分子质量与等电点分别为45.4 ku、4.63。PbSFBB13-gamma表现出典型的花粉SFB/SLF基因的基本结构特征,即1个F-box结构域及4个可变区; 在推导氨基酸水平上,其与蔷薇科SFB/SLF基因的相似性为17.8%~97.7%。试验结果充分表明PbSFBB13-gamma应为花粉S基因的候选基因。选取蔷薇科34个SFB/SLF基因的全长氨基酸序列,构建进化树研究基因间的进化关系。结果表明,34个SFB/SLF基因形成2个亚科特异的类群,但不形成种特异的类群,其进化规律与蔷薇科S-Rnase基因一致,即花粉SFB/SLF基因的形成也是在亚科形成之后、种形成之前。研究结果有助于从分子水平上揭示中国白梨的自交不亲和性机制。

Five Chinese White Pear (Pyrus bretschneideri) cultivars, with known S-genotypes, were used in this study. The genomic DNAs were extracted and amplified by PCR with three pairs of primers corresponding to pear SFBB-alpha, SFBB-beta and SFBB-gamma genes, respectively. Results showed that only one primer pair PSFBG-F/PSFBG-R successfully amplified a fragment of approximately 1 300 bp from ‘Jinhua’ (S13S18),‘Jinhuasihao’ (S13S18) and ‘Eli’ (S13S34) corresponding to pear SFBB-gamma gene. This gene was named PbSFBB13-gamma (Pyrus bretschneideri SFBB13-gamma) and deposited under GenBank accession No. EU081892. RT-PCR revealed that the PbSFBB13-gamma was expressed specifically in the pollen grains. The coding region of the PbSFBB13-gamma was 1 191 bp in length encoding 396 amino acids with a predicted molecular weight of 45.4 ku and isoeletric point of 4.63. The SFBB13-gamma displayed the typical structural characterization of SFB/SLF genes, I.e. an F-box motif and four variable regions. At the deduced amino acid level, it shared 17.8% to 97.7% similarities with other SFB/SLFs of rosaceous plants. These characteristics of PbSFBB13-gamma fully demonstrate that it is a candidate of pollen S-gene although its function in gametophytic self-incompatibility (GSI) response is not confirmed yet. Phylogenetic analysis revealed that 34 rosaceous SFB/SLFs were dived into two subfamily-specific groups, but did not further form species-specific subgroup. The evolutionary pattern of SFB/SLFs concurs with that of rosaceous S-Rnases, suggesting that SFB/SLFs occur after divergence of subfamily but before the divergence of species as S-Rnases do in Rosaceae. The present study could provide a scientific base for fully clarifying the mechanism of pear GSI at the molecular level.


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