‘库尔勒香梨’SFBB-γ基因全长的克隆与序列分析
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摘要
【目的】克隆新疆特色梨品种‘库尔勒香梨’花粉SFBB-γ基因全长序列,为梨亚科SFBB基因的功能鉴定提供更多的依据,为进一步探索自交不亲和机制、培育自交亲和品种奠定分子基础。【方法】以新疆轮台国家果树资源圃采集的‘库尔勒香梨’花粉为材料,参考NCBI上已登录的‘库尔勒香梨’SFBB基因(GQ456943)序列,自行设计6对基因特异性引物和8条RACE巢式引物,筛选合适的引物并通过RT-PCR和RACE技术克隆SFBB-γ基因全长,用DNAMAN软件预测其氨基酸序列,并通过ProtParam等在线预测软件对其进行生物信息学分析;选取NCBI上已登录的全部蔷薇科梨亚科SFBB基因以及部分蔷薇科李属自交不亲和SFB基因的全长序列,用Clustal X软件进行多重比对并用MEGA 4.0构建系统进化树。【结果】从自行设计的6对基因特异性引物和8条RACE巢式引物中,筛选出2对基因特异性引物:[B3F (5’-TCATCCTCCACTTGTATC-3’)/B3R (5’-TAAATCCGTCATCGTAG-3’);B6F (5’-ATGTCTCAGGTGCGTGAAAGTG-3’)/B6R (5’-TAAATCCGTCATCGTAG-3’)]和2条RACE巢式引物:[Outer3 (5’-AACTATTGTACCGTTTCTAAAGGATGG-3’)/Inner5 5’-ATGGGTAATGGACTACGATG-3’)],并通过RT-PCR和RACE技术,成功克隆到1个全长为1 227 bp的SFBB-γ基因(GenBank登录中)。该基因包含1个完整的开放阅读框,长度为1 191 bp,编码396个氨基酸。ProtParam等在线软件预测该蛋白为不稳定亲水性非分泌蛋白,主要参与转运和结合等能量代谢并在基质内起连接酶或裂合酶的作用。聚类分析发现,在69个与蔷薇科花粉自交不亲和性有关的基因中,SFBB基因与SFB基因明显地分成2类;在SFBB基因类中,梨属SFBB-α基因和SFBB-β基因聚成一亚类,其中包括苹果的4个SFBB基因和3个SFLB基因,而梨属SFBB-γ基因则单独聚成另一亚类。【结论】获得了‘库尔勒香梨’花粉SFBB-γ基因c DNA全长序列,该基因由1个F-box区和4个可变区(V1、V2、V3、V4)组成,全长1 127 bp,包含1个完整的1 191 bp的ORF,编码396个氨基酸。构建系统发育树发现,苹果的SFBB基因与梨的SFBB基因聚成一类,其中梨属的SFBB-α基因和SFBB-β基因聚成一亚类,SFBB-γ基因则单独聚成另一亚类。
【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.
【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.
引文
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[2]田嘉,曾斌,罗淑萍,李秀根,李疆.‘库尔勒香梨’PsPL基因的克隆与表达分析[J].果树学报,2015,32(6):1012-1019.TIAN Jia,ZENG Bin,LUO Shuping,LI Xiugen,LI Jiang.Cloning and expression analysis of PsPL gene in‘Korla fragrant pear’(Pyrus sinkiangensis Yu)[J].Journal of Fruit Science,2015,32(6):1012-1019.
[3]NASRALLAH J B,NASRALLAH M E.The molecular genetics of self-incompatibility in Brassica[J].Annual Review of Genetics,1989,23(1):121-139.
[4]CERTAL A C,SANCHEZ A M,KOKKO H,BROOTHAERTSW,OLIVEIRA M M,FEIJO J A.S-RNases in apple are expressed in the pistil along the pollen tube growth path[J].Sexual Plant Reproduction,1999,12(2):94-98.
[5]SASSA H,KAKUI H,MINAMIKAWA M.Pollen-expressed F-box gene family and mechanism of S-RNase-based gametophytic self-incompatibility(GSI)in Roseceae[J].Sexual Plant Reproduction,2010,23(1):39-43.
[6]张绍铃,吴巨友,吴俊,齐永杰,高永彬.蔷薇科果树自交不亲和性分子机制研究进展[J].南京农业大学学报,2012,35(5):53-63.ZHANG Shaoling,WU Juyou,WU Jun,QI Yongjie,GAO Yongbin.The study of self-incompatibility molecular mechanism in Rosaceae fruit tree[J].Journal of Nanjing Agricultural University,2012,35(5):53-63.
[7]SASSA H,KAKUI H,MIYAMOTO M,SUZUKI Y,HANADAT,USHIJIMA K,KUSABA M,HIRANO H,KOBA T.S Locus F-box Brothers:multiple and pollen-specific F-box gene with Shaplotype-specific polymorphisms in apple and Japanese pear[J].Genetics,2007,175(4):1869-1881.
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