中国樱桃花粉SFB基因的克隆及其鉴定
摘要
为分离自交亲和的四倍体植物中国樱桃(Prunus. psedocerasus L.)花粉S-决定子基因,本研究采用中国樱桃品种‘泰小红樱’为试材,根据李属不同树种的花粉SFB基因序列设计兼并引物,应用RT-PCR、RACE等技术分离并鉴定了几个花粉SFB基因。结果如下:
1应用RT-PCR技术,从花粉cDNA中分离到了1个中间片段,与扁桃花粉SFB基因高度同源。用3’-RACE分离到1个3’末端序列,与扁桃花粉SFB基因高度同源。分离到3个5’末端序列,分别与扁桃和梅的花粉SFB基因高度同源。
2拼接5’末端序列、RT-PCR中间片段和3’末端序列后,得到了一个全长序列,全长1171bp,开放式阅读框编码375个氨基酸,N端氨基酸序列具有花粉SFB基因的F-box基序,与扁桃花粉SFB基因高度同源,并具备李属花粉SFB基因的序列特征,命名为SFBc,在GenBank上的注册号为EU267943。
3另外两个5’-端序列核酸序列长度均为656bp,编码214个氨基酸,其N端氨基酸序列都具有花粉SFB基因的F-box基序,分别与梅和扁桃的花粉SFB基因高度同源,分别命名为SFBa和SFBb。SFBa和SFBb在GenBank上的注册号分别为EU267941和EU267942。
4基因的进化树表明,中国樱桃花粉SFB基因与李属花粉SFB/SLF位于同一个进化分枝,而苹果属花粉SFB基因与茄科、玄参科的处于另外一个进化枝。
For isolating the pollen S-determinant genes in Chinese cherry (Prunus. psedocerasus L.), which is a self-compatibility tetroploid species, an Chinese cherry cultivar Taixiaohongying was used as metierial. Using degenerate primers designed based on the conserved regions of the reported SLF/SFB genes, several different cDNA clone were isolated by RT-PCR, The results are as follow:
1. One middle segment cDNA clone was isolated from pollen by cDNA RT-PCR., which chosely homologous to those from amond. 3’RACE in combination with RT-PCR allowed isolation of one 3’segment, which chosely homologous to those from amond. 5’RACE combination with RT-PCR allowed isolation of three 5’segments, which chosely homologous to those from amond and Japanese apricot.
2. The middle segment, 3’segment and one of the three 5’segment composed one full-length cDNA , length 1171bp. The deduced ORFs encode a F-box protein, length 375, which chosely homologous to those from amond, named SFB. We conclude that SFBc (EU267943) is the pollen SFB genes in Chinese cherry.
3. The other two 5’segments, length 656bp, whose corresponding gene SFBa and SBFb were specifically expressed in pollen in an S-haplotype specific manner. The deduced ORFs of the the genes encode twe divergent F-box protein. We conclude that SFBa (GeneBank accession number: EU267941) and SFBb (EU267942) are the pollen SFB genes in Chinese cherry.
4. The phylogenetic trees for SFB proteins reported in Solanaceae, Scrophulariaceae and Rosaceae were constructed. It was found that SFB from Chinese cherry were more closely related to those from Prunus (genus of Rosaceae) than those from Malus (genus of Rosaceae), Petunia (a genus of Solanaceae) and Antirrhinum (genus of Scrophulariaceae).
1应用RT-PCR技术,从花粉cDNA中分离到了1个中间片段,与扁桃花粉SFB基因高度同源。用3’-RACE分离到1个3’末端序列,与扁桃花粉SFB基因高度同源。分离到3个5’末端序列,分别与扁桃和梅的花粉SFB基因高度同源。
2拼接5’末端序列、RT-PCR中间片段和3’末端序列后,得到了一个全长序列,全长1171bp,开放式阅读框编码375个氨基酸,N端氨基酸序列具有花粉SFB基因的F-box基序,与扁桃花粉SFB基因高度同源,并具备李属花粉SFB基因的序列特征,命名为SFBc,在GenBank上的注册号为EU267943。
3另外两个5’-端序列核酸序列长度均为656bp,编码214个氨基酸,其N端氨基酸序列都具有花粉SFB基因的F-box基序,分别与梅和扁桃的花粉SFB基因高度同源,分别命名为SFBa和SFBb。SFBa和SFBb在GenBank上的注册号分别为EU267941和EU267942。
4基因的进化树表明,中国樱桃花粉SFB基因与李属花粉SFB/SLF位于同一个进化分枝,而苹果属花粉SFB基因与茄科、玄参科的处于另外一个进化枝。
For isolating the pollen S-determinant genes in Chinese cherry (Prunus. psedocerasus L.), which is a self-compatibility tetroploid species, an Chinese cherry cultivar Taixiaohongying was used as metierial. Using degenerate primers designed based on the conserved regions of the reported SLF/SFB genes, several different cDNA clone were isolated by RT-PCR, The results are as follow:
1. One middle segment cDNA clone was isolated from pollen by cDNA RT-PCR., which chosely homologous to those from amond. 3’RACE in combination with RT-PCR allowed isolation of one 3’segment, which chosely homologous to those from amond. 5’RACE combination with RT-PCR allowed isolation of three 5’segments, which chosely homologous to those from amond and Japanese apricot.
2. The middle segment, 3’segment and one of the three 5’segment composed one full-length cDNA , length 1171bp. The deduced ORFs encode a F-box protein, length 375, which chosely homologous to those from amond, named SFB. We conclude that SFBc (EU267943) is the pollen SFB genes in Chinese cherry.
3. The other two 5’segments, length 656bp, whose corresponding gene SFBa and SBFb were specifically expressed in pollen in an S-haplotype specific manner. The deduced ORFs of the the genes encode twe divergent F-box protein. We conclude that SFBa (GeneBank accession number: EU267941) and SFBb (EU267942) are the pollen SFB genes in Chinese cherry.
4. The phylogenetic trees for SFB proteins reported in Solanaceae, Scrophulariaceae and Rosaceae were constructed. It was found that SFB from Chinese cherry were more closely related to those from Prunus (genus of Rosaceae) than those from Malus (genus of Rosaceae), Petunia (a genus of Solanaceae) and Antirrhinum (genus of Scrophulariaceae).
引文
成建红,李天忠,韩振海,许雪峰.花粉特异F-box基因及其表达产物可能参与的SCF途径.植物生理学通讯. 2005, 41(1): 90-94.
华志明.植物自交不亲和性分子机理研究的一些进展.植物生理学通讯,1999,2:77-82.
孟金陵.植物生殖生物学.北京:科学出版社.1997,pp214-277
任东路,张燕生,薛勇彪.植物科学进展,1998,1:95-106.
张绍铃,平家伸,徐国华,房经贵刘友良.梨自交不亲和及其亲和突变品种花柱内S4 (S4SM)基因的表达与作用的比较.植物学报,2001,43 (11):1172-1178
朱墨,张开春,姜立杰,张晓明.甜樱桃SFB4与SFB4′基因的鉴别.园艺学报2005,32(1):97-100
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Beecher B, Murfett J, McClure BA. RNase1 from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginfolia pollen. Plant Mol Biol, 1998,36:553-563.
Bosˇkovic′RI ,Wolfram B, Tobutt KR, Cerovic′R, Sonneveld T. Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry. Theor Appl Genet 2006, 112: 315–326
Brewbaker JL. Biology of the angiosperm pollen grain. Indian Genent &plantBreed. 1959,19:121-133.
Broothaerts W, Janssens GA, Proost P, et al. cDNA cloning and molecular analysis of twoself-incompatibility alleles from apple. Plant Mol Biol, 1995,27:499-511
Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Reptr 11: 113-116
Clark, KR, Okuley, Collins PD, et al. Sequence variability and developmental expression of S-alleles in self-incompatible and pseudo-self-compatible Petunia. Plant Cell. 1990,2; 815-826.
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Franklin-Tong VE Franklin FC. Self-incompatibility in Brassica. The elusive pollen S gene is identified. Plant Cell, 2000, 12:305-308.
Franklin-Tong VE, Ride JP, Reed N, et al. The self-incompatibility response in Papver rhoeas is mediated by cytosolic free calcium. Plant J, 1993,4:163-177.
Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M,Vazquez-Santana S, Kim S, Phillips TE, Cruz-Garcia F, McClure B: Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana. Nature 2006, 439:805-810.
Golz, J. F., H.-Y. Oh, V. Su, M. Kusaba and E. Newbigin, 2001 Genetic analysis of Nicotiana pollen-part mutants is consistent with the presence of an S-ribonuclease inhibitor at the S locus. Proc.Natl. Acad. Sci. USA 98: 15372–15376.
Gray JE, Glavin TL, Schafer U, et al. Action of the style product of the self-incompatibility gene Nicotiana alata (S-RNase) on in vitro grown pollen tubes. Plant Ce11,1991,3:271-286
Hauck NR, Yamane H, Tao R, Iezzoni AF: Accumulation of nonfunctional S-haplotypes results in the breakdown of gametophyte self-incompatibility in tetraploid Prunus. Genetics 2006, 172: 1191-1198.
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华志明.植物自交不亲和性分子机理研究的一些进展.植物生理学通讯,1999,2:77-82.
孟金陵.植物生殖生物学.北京:科学出版社.1997,pp214-277
任东路,张燕生,薛勇彪.植物科学进展,1998,1:95-106.
张绍铃,平家伸,徐国华,房经贵刘友良.梨自交不亲和及其亲和突变品种花柱内S4 (S4SM)基因的表达与作用的比较.植物学报,2001,43 (11):1172-1178
朱墨,张开春,姜立杰,张晓明.甜樱桃SFB4与SFB4′基因的鉴别.园艺学报2005,32(1):97-100
Ai Y, Singh A, Coleman CE, et al. Self-incompatibility in Petunia inflata: Isolation and characterization of cDNAs encoding three S-allele-associated proteins. Sex. Plant Reprod. 1990,3:130-138.
Anderson MA, Mcafadden Gi, Bernatzky R, et al. Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata. Plant Cell. 1989, 1:483-491
Anderson MA, Cornish EC, Mau SL, et al. Cloning of cDNA for a stylar glycoprotein associated with expressionof self-incompatibility in Nicotiana alata.. Nature, 1986, 321:38-44.
Beecher B, Murfett J, McClure BA. RNase1 from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginfolia pollen. Plant Mol Biol, 1998,36:553-563.
Bosˇkovic′RI ,Wolfram B, Tobutt KR, Cerovic′R, Sonneveld T. Inheritance and interactions of incompatibility alleles in the tetraploid sour cherry. Theor Appl Genet 2006, 112: 315–326
Brewbaker JL. Biology of the angiosperm pollen grain. Indian Genent &plantBreed. 1959,19:121-133.
Broothaerts W, Janssens GA, Proost P, et al. cDNA cloning and molecular analysis of twoself-incompatibility alleles from apple. Plant Mol Biol, 1995,27:499-511
Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Reptr 11: 113-116
Clark, KR, Okuley, Collins PD, et al. Sequence variability and developmental expression of S-alleles in self-incompatible and pseudo-self-compatible Petunia. Plant Cell. 1990,2; 815-826.
Cornish EC, Pettitt JM, Bonig I, et al. Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata. Nature, 1987,326:99-102
de Nettancourt D. Incompatibility in angiosperms. Berlin: Springer-Verlag, 1977.
De Nettancourt, 2001. Incompatibility and incongruity in wild and cultivated plants. (Berlin:Springer-Verlag).
Dodds PN, Clarke AE Newbigin E. A molecular perspective on pollination in flowering plants. Cell, 1996,85:141-144.
Dowd PE, McCubbin AqWang X, et al. Use of Petunia as a model for the study of Solanaceous type self-incompatibility. Ann. Bot. Suppl. A. 2000, 85:87-93.
Entain T, Iwano M, Shiba H, et al. Comparative analysis of the self-incompatibility (S-) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversity. Genes Cells, 2003,8:203-213.
Foote HC, Ride JP, Frankling-Tong VE, et al. Cloning and expression of a distinctive of class of self-incompatibility (S) gene from papaver rhoeas L. Proc Natl Acad Sci USA, 1994, 91:2265-2269.
Franklin FCH, Lawrence MJ, Franklin-Tong VE. Cell and molecular biologyof self-incompatibility in flowing plants. Int Rev Cytol, 1995, 158:1-64.
Franklin-Tong VE, Franklin FCH. Gametophytic self-incompatibility in Papaver rhoeas: There is no evidence for the involvement of stigmatic ribonuclease ability. Plant Cell Enviro,1991,14:423-429.
Franklin-Tong VE Franklin FC. Self-incompatibility in Brassica. The elusive pollen S gene is identified. Plant Cell, 2000, 12:305-308.
Franklin-Tong VE, Ride JP, Reed N, et al. The self-incompatibility response in Papver rhoeas is mediated by cytosolic free calcium. Plant J, 1993,4:163-177.
Goldraij A, Kondo K, Lee CB, Hancock CN, Sivaguru M,Vazquez-Santana S, Kim S, Phillips TE, Cruz-Garcia F, McClure B: Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana. Nature 2006, 439:805-810.
Golz, J. F., H.-Y. Oh, V. Su, M. Kusaba and E. Newbigin, 2001 Genetic analysis of Nicotiana pollen-part mutants is consistent with the presence of an S-ribonuclease inhibitor at the S locus. Proc.Natl. Acad. Sci. USA 98: 15372–15376.
Gray JE, Glavin TL, Schafer U, et al. Action of the style product of the self-incompatibility gene Nicotiana alata (S-RNase) on in vitro grown pollen tubes. Plant Ce11,1991,3:271-286
Hauck NR, Yamane H, Tao R, Iezzoni AF: Accumulation of nonfunctional S-haplotypes results in the breakdown of gametophyte self-incompatibility in tetraploid Prunus. Genetics 2006, 172: 1191-1198.
Hearn MJ, Franklin FCH, Ride JP. Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility (S-) protein. Plant J, 1996,9:467-475.
Hecht A. 1944. Induced tetraploids of a self sterile Oenothera. Genetics 29: 69–74.
Hiscock SJ, Kues U, Dichinson HG. Molecular mechanisms of self-incompatibility in flowering plants and fungi-different means to the same end. Trends Cell Biol, 1996,6:421-428
Huang S, Lee H-S, Karunanandaa B, et al. Ribonuclease activity of Petuniainflata S proteins is essential for rejection of self-pollen. Plant Cell.,1994, 6: 1021一1028.
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