柠檬F-box基因的克隆及表达分析
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摘要
本研究以香水柠檬幼嫩叶片为材料,在前期通过De Novo技术获得的F-box基因片段基础上设计引物,利用RT-PCR技术成功克隆出长度为585 bp的F-box基因,命名为LFB1,该基因编码194个氨基酸,该氨基酸序列在N端具有F-box结构域,生物信息学分析表明:该蛋白分子量为48.94 k D,等电点:5.2。进化分析发现其与克里曼丁桔(Citrus clementina)聚在一起。q RT-PCR分析表明:该基因在植株的根、茎、嫩叶、老叶、花蕾、花苞、幼果、成熟果中均有表达,LFB1基因在组织中的表达量是:茎>老叶>根>花蕾>成熟果>嫩叶>幼果;在花的不同器官的表达分析,发现该基因在雄蕊中的表达量最高,其它组织则相对较低,在雄蕊中基因的表达量是其它组织的10倍左右;在逆境胁迫下,该基因的表达量发生明显变化,表明该基因与植株的生长发育、花粉的发育以及环境胁迫可能有着一定的关系。
A length o f 585 bp F-box gene, named LFB1, was successfully cloned from ‘Xiangshui'lemon tender leaves by RT-PCR methods, which encoded 194 amino acids, the amino acid sequence has F-box structure. Bioinformatics analysis showed that the molecular weight of the protein was 48.94 k D, its isoelectric point was 5.2; Phylogenetic analysis revealed that it clustered with Citrus clementina. q RT-PCR analysis showed that the gene expressed in plant roots, stems, leaves, leaflets, bud, bud, young fruit and mature fruit,The mount of LFB1 gene expression in tissue was: stem>old leaves>root>bud>fruit>mature leaves>young fruit.In the The expression results in different flower organ, indicated that the gene was expressed highest in the stamens, other groups expressed relatively low, the quantity of gene expression in the stamens was about 10 times as much as other organizations. Under adversity stress, the gene expression of the quantity change obviously, the results showed that the gene had a relationship with the pollen germination, growth and environmental stress.
A length o f 585 bp F-box gene, named LFB1, was successfully cloned from ‘Xiangshui'lemon tender leaves by RT-PCR methods, which encoded 194 amino acids, the amino acid sequence has F-box structure. Bioinformatics analysis showed that the molecular weight of the protein was 48.94 k D, its isoelectric point was 5.2; Phylogenetic analysis revealed that it clustered with Citrus clementina. q RT-PCR analysis showed that the gene expressed in plant roots, stems, leaves, leaflets, bud, bud, young fruit and mature fruit,The mount of LFB1 gene expression in tissue was: stem>old leaves>root>bud>fruit>mature leaves>young fruit.In the The expression results in different flower organ, indicated that the gene was expressed highest in the stamens, other groups expressed relatively low, the quantity of gene expression in the stamens was about 10 times as much as other organizations. Under adversity stress, the gene expression of the quantity change obviously, the results showed that the gene had a relationship with the pollen germination, growth and environmental stress.
引文
An Z.Y.,He X.H.,D ong L.,Zhang S.W.,Hu Y.,Luo C.,and Huang G.X.,2015,Studies on extraction meth ods of to tal RNA from lemon tiss ues,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):203-207(安振宇,何新华,董龙,张树伟,胡颖,罗聪,黄桂香,2015,柠檬总RNA提取方法研究,基因组学与应用生物学,34(1):203-207)
Bai C.,Sen P.,Hofmann K.,Ma L.,Goebl M.,Harper J.W.,and Elledge S.J.,1996,SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif,the F-box,Cell,86(2):263-274
Cheng J.H.,Li T.Z.,Han Z.H.,and Xu X.F.,2005,Pollen specific F-box gene and its expressed product involved in putative SCF pathway,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(1):90-94(成建红,李天忠,韩振海,许雪峰,2005,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,41(1):90-94)
Cheng J.H.,Bai S.L.,Han Z.H.,Xu X.F.,and Li T.Z.,2006,Cloning and expression analysis of the self-incompatibility S9-Haplotype specific F-box genes in cherry(Prunus avium),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(5):976-983(成建红,白松龄,韩振海,许雪峰,李天忠,2006,樱桃自交不亲和S9-单元型特异的F-box基因克隆及其表达分析,中国农业科学,39(5):976-983)
Cheng J.H.,Han Z.H.,Xu X.F.,and Li T.Z.,2006,Isolation and identification of the pollen-expressed polymorphic F-box genes linked to the S-lous in apple(Malus×domestica),Sex.Plant Reprod.,19(4):175-183
Deshaies R.J.,1999,SCF and Cullin/Ring H2-based ubiquitin ligases,Annu.Rev.Cell Dev.Biol.,15(1):435-467
Gonzalez Z.H.,Rompa U.,Peters J.L.,Bhatt A.M,Wagstaff C.,Stead A.D.,and Roberts J.A.,2007,Hawaiian skirt:an F-box gene that regulates organ fusion and growth in Arabidopsis,Plant Physiol.,144(3):1370-1382
Kumar A.,and Paietta J.V.,1995,The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats,Proc.Natl.Acad.Sci.USA,92(8):3343-3347
Luo C.,2012,Study on SCo T marker and analysis on genes of stress-related and important flowering time in mango,Dissertation for Ph.D.,College of Agriculture,Guangxi Univer sity,Supervisor:He X.H.,pp.1-238(罗聪,2012,芒果SCo T分子标记与逆境和重要开花时间相关基因研究,博士学位论文,广西大学农学院,导师:何新华,pp.1-238)
Luo C.,He X.H.,Chen H.,Wei Y.L.,and Li M.J.,2011,Ahigh-efficient method of RACE technique for obtaining the gene 5'end,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(4):409-414(罗聪,何新华,陈虎,韦泳丽,李明娟,2011,一种高效获取基因5'末端的RACE方法,植物生理学报,47(4):409-414)
Lechner E.,Achard P.,Vansiri A.,Potuschak T.,and Genschik P.,2006,F-box proteins everywhere,Curr.Opin.Plant Biol.,9(6):631-638
Li L.,Li Y.X.,Xia K.,Song S.F.,and Zhou Z.W.,2010,Plant F-Box proteins and their biological functions,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),38(35):19879-19881(李莉,李懿星,夏凯,宋书锋,周在为,2010,植物F-Box蛋白及其生物学功能研究,安徽农业科学,38(35):19879-19881)
Mc Cubbin A.G.,and Kao T.H.,2000,Molecular recognition and response in pollen and pistil interactions,Annu.Rev.cell Dev.Biol.,16:333-364
Sassa H.,Kakui H.,Miyamoto M.,Suzuki Y.,Hanada T.,Ushijima K.,Kusaba M.,Hirano H.,and Koba T.,2007,S locus F-Box brothers:multiple and pollen-specific F-Box genes with S haplotype-specific polymorphisms in apple and japanese pear,Genetics,175(4):1869-1881
Schulman B.A.,Carrano A.C.,Jeffrey P.D.,Bowen Z.,Kinnucan E.R.E.,Finnin M.S.,Elledge S.J.,Harper J.W.,Pagano M.,and Pavletich N.P.,2000,Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex,Nature,408(6810):381-386
Seagull R.W.,and Giavalis S.,2004,Pre-and post-anthesis application of exogenous hormones alters fiber production in Gossypium hirsutum L.cultivar Maxxa GTO,J.Cotton Science,8:105-111
Smalle J.,Kurepa J.,Yang P.,Emborg T.J.,Babyichuk E.,Kushnir S.,and Vierstra R.D.,2003,The pleiotropic role of the26S proteasome subunit RPN10 in Arabidopsis thaliana growth and development supports a substrate-specific function in abscisic acid signaling,The Plant Cell,15(4):965-980
Smalle J.,and Vierstra R.D.,2004,The ubiquitin 26S proteasome proteolytic pathway,Plant Biol.,55:555-590
Wei J.H.,Fan S.L.,Song M.Z.,Pang C.Y.,and Yu S.X.,2011,Cloning and expression analysis of Gh FBO in Gossypium hirsutum L.,Mianhua Xuebao(Cotton Science),23(3):212-218(卫江辉,范术丽,宋美珍,庞朝友,喻树迅,2011,棉花一个新F-Box基因的克隆与表达分析,棉花学报,23(3):212-218)
Wu J.,Peng X.Z.,Yuan J.G.,and Qiang B.Q.,2002,Progress in functional characterization of F-box protein family,Shengwu Huaxue Yu Shengwu Wuli Jinzhan(Progress in Biochemistry and Biophysics),29(4):503-506(吴静,彭小忠,袁建刚,强伯勤,2002,F-box蛋自家族的功能研究进展,生物化学与生物物理进展,29(4):503-506)
Xing L.,and Li Z G..,2011,Expression analysis of a F-box gene in tomato and construction of it's plant expression vectors,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(4):658-662(邢蕾,李正国,2011,番茄中一个F-box/FBA基因的表达分析及载体构建,热带作物学报,32(4):658-662)
Zhu K.K.,Zhang C.F.,Yang F.,Han Z.H.,and Li T.Z.,2009,Cloning and identification of pollen F-box gene Md SLFB9in apple(Malus×domestica),Xibei Zhiwu Xuebao(Acta B-otanica Boreali-Occidentalia Sinica),29(4):650-655(朱开开,张春芳,杨锋,韩振海,李天忠,2009,苹果花粉F-box基因Md SLFB9克隆与鉴定,西北植物学报,29(4):650-655)
Bai C.,Sen P.,Hofmann K.,Ma L.,Goebl M.,Harper J.W.,and Elledge S.J.,1996,SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif,the F-box,Cell,86(2):263-274
Cheng J.H.,Li T.Z.,Han Z.H.,and Xu X.F.,2005,Pollen specific F-box gene and its expressed product involved in putative SCF pathway,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(1):90-94(成建红,李天忠,韩振海,许雪峰,2005,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,41(1):90-94)
Cheng J.H.,Bai S.L.,Han Z.H.,Xu X.F.,and Li T.Z.,2006,Cloning and expression analysis of the self-incompatibility S9-Haplotype specific F-box genes in cherry(Prunus avium),Zhongguo Nongye Kexue(Scientia Agricultura Sinica),39(5):976-983(成建红,白松龄,韩振海,许雪峰,李天忠,2006,樱桃自交不亲和S9-单元型特异的F-box基因克隆及其表达分析,中国农业科学,39(5):976-983)
Cheng J.H.,Han Z.H.,Xu X.F.,and Li T.Z.,2006,Isolation and identification of the pollen-expressed polymorphic F-box genes linked to the S-lous in apple(Malus×domestica),Sex.Plant Reprod.,19(4):175-183
Deshaies R.J.,1999,SCF and Cullin/Ring H2-based ubiquitin ligases,Annu.Rev.Cell Dev.Biol.,15(1):435-467
Gonzalez Z.H.,Rompa U.,Peters J.L.,Bhatt A.M,Wagstaff C.,Stead A.D.,and Roberts J.A.,2007,Hawaiian skirt:an F-box gene that regulates organ fusion and growth in Arabidopsis,Plant Physiol.,144(3):1370-1382
Kumar A.,and Paietta J.V.,1995,The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats,Proc.Natl.Acad.Sci.USA,92(8):3343-3347
Luo C.,2012,Study on SCo T marker and analysis on genes of stress-related and important flowering time in mango,Dissertation for Ph.D.,College of Agriculture,Guangxi Univer sity,Supervisor:He X.H.,pp.1-238(罗聪,2012,芒果SCo T分子标记与逆境和重要开花时间相关基因研究,博士学位论文,广西大学农学院,导师:何新华,pp.1-238)
Luo C.,He X.H.,Chen H.,Wei Y.L.,and Li M.J.,2011,Ahigh-efficient method of RACE technique for obtaining the gene 5'end,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(4):409-414(罗聪,何新华,陈虎,韦泳丽,李明娟,2011,一种高效获取基因5'末端的RACE方法,植物生理学报,47(4):409-414)
Lechner E.,Achard P.,Vansiri A.,Potuschak T.,and Genschik P.,2006,F-box proteins everywhere,Curr.Opin.Plant Biol.,9(6):631-638
Li L.,Li Y.X.,Xia K.,Song S.F.,and Zhou Z.W.,2010,Plant F-Box proteins and their biological functions,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),38(35):19879-19881(李莉,李懿星,夏凯,宋书锋,周在为,2010,植物F-Box蛋白及其生物学功能研究,安徽农业科学,38(35):19879-19881)
Mc Cubbin A.G.,and Kao T.H.,2000,Molecular recognition and response in pollen and pistil interactions,Annu.Rev.cell Dev.Biol.,16:333-364
Sassa H.,Kakui H.,Miyamoto M.,Suzuki Y.,Hanada T.,Ushijima K.,Kusaba M.,Hirano H.,and Koba T.,2007,S locus F-Box brothers:multiple and pollen-specific F-Box genes with S haplotype-specific polymorphisms in apple and japanese pear,Genetics,175(4):1869-1881
Schulman B.A.,Carrano A.C.,Jeffrey P.D.,Bowen Z.,Kinnucan E.R.E.,Finnin M.S.,Elledge S.J.,Harper J.W.,Pagano M.,and Pavletich N.P.,2000,Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex,Nature,408(6810):381-386
Seagull R.W.,and Giavalis S.,2004,Pre-and post-anthesis application of exogenous hormones alters fiber production in Gossypium hirsutum L.cultivar Maxxa GTO,J.Cotton Science,8:105-111
Smalle J.,Kurepa J.,Yang P.,Emborg T.J.,Babyichuk E.,Kushnir S.,and Vierstra R.D.,2003,The pleiotropic role of the26S proteasome subunit RPN10 in Arabidopsis thaliana growth and development supports a substrate-specific function in abscisic acid signaling,The Plant Cell,15(4):965-980
Smalle J.,and Vierstra R.D.,2004,The ubiquitin 26S proteasome proteolytic pathway,Plant Biol.,55:555-590
Wei J.H.,Fan S.L.,Song M.Z.,Pang C.Y.,and Yu S.X.,2011,Cloning and expression analysis of Gh FBO in Gossypium hirsutum L.,Mianhua Xuebao(Cotton Science),23(3):212-218(卫江辉,范术丽,宋美珍,庞朝友,喻树迅,2011,棉花一个新F-Box基因的克隆与表达分析,棉花学报,23(3):212-218)
Wu J.,Peng X.Z.,Yuan J.G.,and Qiang B.Q.,2002,Progress in functional characterization of F-box protein family,Shengwu Huaxue Yu Shengwu Wuli Jinzhan(Progress in Biochemistry and Biophysics),29(4):503-506(吴静,彭小忠,袁建刚,强伯勤,2002,F-box蛋自家族的功能研究进展,生物化学与生物物理进展,29(4):503-506)
Xing L.,and Li Z G..,2011,Expression analysis of a F-box gene in tomato and construction of it's plant expression vectors,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(4):658-662(邢蕾,李正国,2011,番茄中一个F-box/FBA基因的表达分析及载体构建,热带作物学报,32(4):658-662)
Zhu K.K.,Zhang C.F.,Yang F.,Han Z.H.,and Li T.Z.,2009,Cloning and identification of pollen F-box gene Md SLFB9in apple(Malus×domestica),Xibei Zhiwu Xuebao(Acta B-otanica Boreali-Occidentalia Sinica),29(4):650-655(朱开开,张春芳,杨锋,韩振海,李天忠,2009,苹果花粉F-box基因Md SLFB9克隆与鉴定,西北植物学报,29(4):650-655)