梨极矮化突变体S基因型鉴定与DELLA蛋白编码基因的克隆
|
摘要
本研究以延边小香水梨实生后代中发现的矮化紧凑的突变体等17个梨属砧木及延边地区栽培梨品种为试材,应用PCR扩增及克隆、测序技术分离其S基因,使用生物信息学软件对各序列进行分析和同源性搜索比对,最终确定各品种的S基因型,初步推测梨极矮化突变体亲本；应用RT-PCR、cDNA末端快速扩增技术(RACE)克隆梨极矮化突变体和苹果梨DELLA家族蛋白编码基因的编码区全长。主要研究结果如下：
梨矮化突变体等17个梨属砧木及延边地区栽培梨品种的S基因型分别为：“极矮化突变体”、“苹果梨”、“东宁五号大梨”、“延光梨”、“S2(中矮一号)”、“S7”为S1S17；“苹博香”为S1S8m；“早酥”为SlSd；“延边谢花甜”、“S5”为S17S31；“尖把梨”为S12S30；“延边明月梨”为S3Se；“小香水芽变”为SeSx；“朝鲜洋梨”为SeS3；“身不知”为S5Sd；“PDR54(中矮二号)”为。S4S8S17；“山梨”为S12Sx。其中“苹博香”的S8m为新基因,“PDR54”为三倍体。
利用RACE技术从梨极矮化突变体中克隆到一个编码DELLA家族蛋白的全长cDNA序列,命名为PuRGL。该基因全长1743bp,包含一个完整的编码580个氨基酸的开放阅读框(ORF).PuRGL与MdRGL2α.MhGAI、RcGAI、PtRGAS.AtRGL2的同源性分别为98%、98%、82%、81%、77%；结构分析表明该基因具有DELLA家族蛋白的典型结构域。
利用RACE技术从苹果梨中克隆到两个编码DELLA家族蛋白的全长cDNA序列,分别命名为PbRGLl和PbRGL2。PbRGL1全长1755bp,包含一个完整的编码584个氨基酸的ORF,PbRGL1与MdRGL2b、MhGAI、RcGAI、PtGAS、AtRGL2的同源性分别为99%、93%、83%、82%、78%、78%、PbRGL2全长1743bp,包含一个完整的编码580个氨基酸的ORF, PbRGL2与MdRGL2α、MhGAI、RcGAI、PtGRAs、AtRGL2的同源性分别为98%、98%、82%、81%、77%。结构分析表明以上两个基因具有DELLA家族蛋白的典型结构域。
This experiment with estimation of chance seedling dwarf mutant of Yanbian xiaoxiangshuili (Pyrus.ussuriensis Maxim) and other sixteen Yanbian pear cultivars, pear rootstocks as the materials, Using a pair of primers specific to pear S-RNases, PCR amplification from their genomic DNAs. Then the PCR products were extracted, cloned and sequenced. The amplified fragments were assigned to their respective S-RNases by blast analysis, conjecture the parents of pear dwarf mutant; RT-PCR and Rapid Amplification of cDNA Ends(RACE) technique was used to obtain a full length cDNA of the gene encoding DELLA protein. the result are as follows:
The S-genotypes of the pear dwarf mutant and other sixteen Yanbian pear cultivars, pear rootstocks were identified as follows: 'dwarf mutant '(S1S17); 'Pingguoli' (S1S17); 'Yanguangli'(S1S17);'Dongning5haodali'(S1S17);'Pingboxiang'(S1S8m);'Zaosu'(S1Sd);'Yanbianxiehuatian '(S17S31);'Jianbali'(S12S30);'Yanbianmingyueli'(S3Se);'Xiaoxiangshuiyabian'(SeSx);'Chaoxianyangli'(SeS x);'Mishirazu'(S5Sx);'Shanli'(S12Sx);'S2'(S1S17);'S5'(S17S31);'S7'(S1S17);'PDR54 (S4S8S17).The S8m of 'Pingboxiang' is new gene,'PDR54'was triploid plant.
A gene encoding DELLA protein was isolated from pear dwarf mutant by RACE technique,named PuRGL. It is 1743 bp, coding a polypeptide of 580 amino acids. Homology analysis showed that the deduced PuRGL protein was highly homologous o the MdRGL2a、MhGAI、RcGAI、PtRGAS、AtRGL2, the identity respectively is 98%、98%、82%、81%、77%.
Two genes encoding DELLA protein was isolated from Pingguoli by RACE technique.The one gene is long 1755 bp, coding a polypeptide of 584 amino acids,named PaRGL1. Homology analysis showed that the deduced PaRGL1 protein was highly homologous to the MdRGL2b、MhGAI、RcGAI、PtGAS、AtRGL2, the identity respectively is 99%、93%、83%、82%、78%、78%;Another gene is long 1743 bp, coding a polypeptide of 580 amino acids,named PaRGL2. Homology analysis showed that the deduced PaRGL2 protein was highly homologous to the MdRGL2α、MhGAI、RcGAI、PtGRAS、AtRGL2, the identity respectively is 98%、98%、82%、81%、77%.
梨矮化突变体等17个梨属砧木及延边地区栽培梨品种的S基因型分别为：“极矮化突变体”、“苹果梨”、“东宁五号大梨”、“延光梨”、“S2(中矮一号)”、“S7”为S1S17；“苹博香”为S1S8m；“早酥”为SlSd；“延边谢花甜”、“S5”为S17S31；“尖把梨”为S12S30；“延边明月梨”为S3Se；“小香水芽变”为SeSx；“朝鲜洋梨”为SeS3；“身不知”为S5Sd；“PDR54(中矮二号)”为。S4S8S17；“山梨”为S12Sx。其中“苹博香”的S8m为新基因,“PDR54”为三倍体。
利用RACE技术从梨极矮化突变体中克隆到一个编码DELLA家族蛋白的全长cDNA序列,命名为PuRGL。该基因全长1743bp,包含一个完整的编码580个氨基酸的开放阅读框(ORF).PuRGL与MdRGL2α.MhGAI、RcGAI、PtRGAS.AtRGL2的同源性分别为98%、98%、82%、81%、77%；结构分析表明该基因具有DELLA家族蛋白的典型结构域。
利用RACE技术从苹果梨中克隆到两个编码DELLA家族蛋白的全长cDNA序列,分别命名为PbRGLl和PbRGL2。PbRGL1全长1755bp,包含一个完整的编码584个氨基酸的ORF,PbRGL1与MdRGL2b、MhGAI、RcGAI、PtGAS、AtRGL2的同源性分别为99%、93%、83%、82%、78%、78%、PbRGL2全长1743bp,包含一个完整的编码580个氨基酸的ORF, PbRGL2与MdRGL2α、MhGAI、RcGAI、PtGRAs、AtRGL2的同源性分别为98%、98%、82%、81%、77%。结构分析表明以上两个基因具有DELLA家族蛋白的典型结构域。
This experiment with estimation of chance seedling dwarf mutant of Yanbian xiaoxiangshuili (Pyrus.ussuriensis Maxim) and other sixteen Yanbian pear cultivars, pear rootstocks as the materials, Using a pair of primers specific to pear S-RNases, PCR amplification from their genomic DNAs. Then the PCR products were extracted, cloned and sequenced. The amplified fragments were assigned to their respective S-RNases by blast analysis, conjecture the parents of pear dwarf mutant; RT-PCR and Rapid Amplification of cDNA Ends(RACE) technique was used to obtain a full length cDNA of the gene encoding DELLA protein. the result are as follows:
The S-genotypes of the pear dwarf mutant and other sixteen Yanbian pear cultivars, pear rootstocks were identified as follows: 'dwarf mutant '(S1S17); 'Pingguoli' (S1S17); 'Yanguangli'(S1S17);'Dongning5haodali'(S1S17);'Pingboxiang'(S1S8m);'Zaosu'(S1Sd);'Yanbianxiehuatian '(S17S31);'Jianbali'(S12S30);'Yanbianmingyueli'(S3Se);'Xiaoxiangshuiyabian'(SeSx);'Chaoxianyangli'(SeS x);'Mishirazu'(S5Sx);'Shanli'(S12Sx);'S2'(S1S17);'S5'(S17S31);'S7'(S1S17);'PDR54 (S4S8S17).The S8m of 'Pingboxiang' is new gene,'PDR54'was triploid plant.
A gene encoding DELLA protein was isolated from pear dwarf mutant by RACE technique,named PuRGL. It is 1743 bp, coding a polypeptide of 580 amino acids. Homology analysis showed that the deduced PuRGL protein was highly homologous o the MdRGL2a、MhGAI、RcGAI、PtRGAS、AtRGL2, the identity respectively is 98%、98%、82%、81%、77%.
Two genes encoding DELLA protein was isolated from Pingguoli by RACE technique.The one gene is long 1755 bp, coding a polypeptide of 584 amino acids,named PaRGL1. Homology analysis showed that the deduced PaRGL1 protein was highly homologous to the MdRGL2b、MhGAI、RcGAI、PtGAS、AtRGL2, the identity respectively is 99%、93%、83%、82%、78%、78%;Another gene is long 1743 bp, coding a polypeptide of 580 amino acids,named PaRGL2. Homology analysis showed that the deduced PaRGL2 protein was highly homologous to the MdRGL2α、MhGAI、RcGAI、PtGRAS、AtRGL2, the identity respectively is 98%、98%、82%、81%、77%.
引文
[1]Reid J B. Plant hormone mutants[J]. Journal of Plant Growth Regulation,1993, 12:207-226.
[2]毕晓颖,吴禄平,安利佳.一个与苹果属显性矮生主基因Dw连锁的RAPD标记[J].园艺学报2002,29(1)：1-4.
[3]张所兵.水稻半矮杆突变体株高遗传分析及eui基因定位[D].南京：南京农业大学,2006年6月.
[4]张达,王军虹,王豫颖,等.矮化大豆突变体GA3调控[J].大豆科学,2008,37(3)：456-460.
[5]朴永虎.延边小香水梨(Pyrus. ussuriensis Maxim)偶然实生矮化突变体的评价研究[D]. 延吉：延边大学硕士论文,2010年6月.
[6]孟金陵.植物生殖遗传学[M].北京：科学出版社,1995.
[7]Lewis D.Incompatibility in floiwng plants[J]. Recept Recognition Ser,1976,A2:167-198.
[8]张绍铃,平伸,徐国华,等.梨自交不亲和及其突变品种花柱内S4和S4SM基因的表达与作用比较[J].植物学报,2001,43(11)：1172-1178.
[9]辜青青.利用PCR-RFLP技术鉴定部分沙梨(Pyrus pyrifolia Nakai)品种S基因型[D].武汉：华中农业大学硕士论文,2006,7-8.
[10]Brewbaker J L.Polen Cytology and Self-incompatibility Systems in Plants[J].Hered,1957,48:271-277.
[11]Vaughan S P,Russel L K,Sargent D J, et al.Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype[J].Theor Appl Genet 2006,112:856-866.
[12]Sylvain B,Vincent C,Xavier V.A general model to explore complex dominance patterns in plant sporophytic self-incompatibility systems[J].Genetics,2007.175:1351-1369.
[13]王茂广.孢子体型自交不亲和反应臂重复蛋白ARC1[J].生命科学,2007,19(1)：86-90.
[14]Kikuchi A. Investigation in 1927 and 1928, Pater clinical incompatibility in the Japanese pear [J].Okitse.hort.Soc.1929,24:1-6.
[15]Ushikoshi M, Tokuyasu K. Studies on pollination and fertilization in pear [J]. Okitse. Hort. Soc,1930,25:135-152.
[16]张绍铃,曹生民,吴华清.果树自交不亲和性基因型及其鉴定方法[J].果树学报,2003,20(05)：358-363.
[17]曾艳玲.鹅梨S基因型的鉴定及S基因的cDNA克隆[D].长沙：中南林业科技大学,2006.
[18]乌云塔娜,谭晓风,曹玉芬,等.白梨品种4个新S基因的分离与鉴定[J].园艺学报,2007,34(04)：859-864.
[19]乌云塔娜,谭晓风,毕方铖,等.中国白梨S基因研究Ⅰ：7个品种S基因型的确定和2个新S基因的鉴定[J].中南林学院学报,2005,25(04)：7-12.
[20]谭晓风,毕方铖,乌云塔娜.中国白梨S基因研究Ⅱ：9个主栽品种S基因型的确定及S29-RNase新基因的分离鉴定[J].中南林学院学报,2005,25(04)： 13-16.
[21]袁德义,谭晓风,张琳,等.新高系梨10个品种S基因型的鉴定[J].园艺学报,2007,34(6)：1353-1360.
[22]Ishimizu T, Inoue K, Shimonaka M, et al. PCR-based method for identifying the S-genotypes of Japanese pear cultivars [J].Theor Appl Genet,1999,98:961-967
[23]张春芳,李茂福,韩振海,等.秋子梨等九个品种S基因型的鉴定[J].北方园艺,2008(11)：131—134.
[24]衡伟,张绍铃,张妤艳,等.12个梨品种S基因型的鉴定[J].园艺学报,2007,34(04)：853-858.
[25]Nrioka N,Norilka S,Ohnishi Y. Molecular cloning and nucleotide sequenece of cDNA encoding S-allele specific stylar RNases in a self-incompatible cultivar and its self-compatible mutant of Japanese pear[J]. J. Biochem.,1996, (120):335-345.
[26][美]谢纳.生物芯片分析[M].北京：科学出版社,2004,86-227.
[27]虞慧芳,曹家树,王永勤.植物矮化突变体的激素调控[J].生命科学,2002,14(2)：85-88.
[28]Fu X,Richards DE,Air-ali T et al,Gibberellin-mediated proteasome-dependent degradation of the barley DELLAprotein [J].SLNI repressorPlant Cell,2002,14:3191-3200.
[29]Frohman M A, Dush M K, Mrtin G R. Rapid production of full- length cDNAs from rare transcripts: amplification using single gene -specific oligonuleotide primer[J]. Proc. Natl. Acad. Sci. USA,1988,85(23):8998-9002.
[30]杨铁钊,杨欣玲,殷全玉,等.烟草株高变异体的茎尖内源激素含量变化及其对外源激素的响应[J].植物生理学通讯,2006,42(4)：643-647.
[31]Hedden P, Phillips A L. Gibberellin metabolism: new insights revealed by the genes[J]. Trends in Ptant Science,2000,5(12):523-530.
[32]Ohara O, Dorit R L, Gillbert W. One-sided polymerase chain reaction: the amplification of Cdna[J]. Proc. Natl. Acad. Sci. USA,1989,86(15):5673-5677.
[33]Loh E Y, Elliot J F, Cwirla S, Lanier L L,Davis M M. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain[J]. Science, 1989,243 (4888):217-220.
[34]Schaefer B C. Resolution in rapid amplification of cDNA ends: New strategies for polymerase chain reaction cloning of full-length C dna ends[J]. Anal. Biochem.,1995,227:255-273.
[35]王月华,韩烈保,曾会明,等.植物赤霉素矮化突变体研究进展术[J].中国生物工程杂志,2006,26(8)：22-27.
[36]伍涛,曹家树,虞慧芳.赤霉素合成基因的克隆以及其相关矮化突变体[J].细胞生物学杂志,2005,27：157-164.
[37]Dill A, Jung HS, Sun TP. The DELLA motif is essential for gibberellin-induced degradation of RGA[J]. Proceedings of the National Academy of Sciences,2001, 98:14162-14167.
[38]Pysh L D,Wysocka-Diller J W,Camilleri C,Bouchez, et al.The GRAS family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes [J].Plant J.18,111-119.
[39]Peng J,Carol P,Richards DE,et al.The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses[J].GenesDev,1997,11:3194-8205.
[40]Peng J,Richards D E,Hartley N M, et al.'Green revolution'genes encode mutant gibberellin response modulators[J].Nature,1999,400,256-261.
[41]Chandler PM,Poll AM,Ellis M et al.Mutants at the Sienderl locus of barley Himalaya[J].Molecular and physiological characterization.PlutPhysiol,2002,129:181-190.
[42]Boss P K,Thomas M R. Association of dwarfism and floral in duction with a grape 'green revolution'mutation[J].Nature,2000.1416:847-850.
[43]Ikeda A,Ueguchi-Tanaka M,Sonoda Y,et al.Slender rice,aconstimtive gibberellin response mutant,is caused by a null mutation of the SLRl gene.an ortholog of the height regulating gene,GAI/RGA/RHT/D8[J].Plant Cell,2001.13:999-1010.
[44]Lee S,Cheng H,Peng J, et al.Gibberelin regulates Arabidopsis seed germination via RGL2,a GAI/RGA-like gene whose expression is up-regulated folowing imbibition[J]. Genes Dev,2002,16:646-658.
[45]Ogawa M,Kusano T,Sano H et al.Rice gibberellin-insensitive gene homolog,OsGAI, encodes a nuclear localized protein Capable of gene activation at transcriptional level [J].Gene,2002,45:21-29.
[46]Itoh H,Uegechi-Tanaka M,Sato Y,et al. The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE 1 in nuclei[J]. Plant Cell,2002,14:57-70.
[47]黄先忠,马正强.DELLA家族蛋白与植物生长发育的关系[J].植物生理学通讯,2004.40(5)：529-532.
[48]Salchert K, Bhalerao R, Koncz-Kalman Z, Koncz C. Control of cell elongation and stress responses by steroid hormones and carbon catabolic repression in plants[J]. Series B, Biological Sciences,1998,353(1374):1517-1520.
[49]Chory J, Nagpal P, Peto C A. Phenotypic and gnetic analysis of det2, a new mutant that affects light-regulated seedling development in Arabidopsis[J]. The Plant Cell,1991,3:445-459.
[50]Kauschmann A,Jessop A,Koncz C,et al. Genetic evidence for an essential role of brassiriosteroids in plant development[J].Plant J,1996.9:701-713.
[51]Friedrichaen D M, Joazeiro, Hunter T,Chory J. Brassinosteroid-insensitive-I is a ubiquitously excpressed leucine-rich repeat roceptor serine/threonine kinase[J]. Plant Physiology,2000,123:1247-1255.
[52]Yang T, Davies PJ, Reid JB. Genetic dissection of the relative roles of auxin and gibberellin in the regulation of stem elongation in intact light-grnwn peas[J]. Plant Physiology,1996,110:1029-1034.
[53]Sato Y, Sentoku N, Miura Y, Hirochika H, Kitano H, Matsuoka M. Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants[J]. The European Molecular Biology Organization Journal,1999,18(4):992-1002.
[54]Kusaba S, Fukumoto M, Honda C, Yamaguchi I, Sakamoto T, Kano M Y Decreased GAi content caused bythe overoxprossion of OSH1 is acoompanied by suppression of GA20-oxidaae geneexpression[J].Plant Physiology,1998.117:1179-1184.
[55]Tsiantis M, Brown M I N, Skibinski G.. Disruption of auxin transport is associated with aberrant leaf development in maize[J]. Plant Physiologx 1999, 121(4):1163-1168.
[56]Reiter W D, Chapple C C S, Somerville C R. Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis[J]. Science,1993,261:1032-1035.
[57]Weyers J DB, Paterson N W, Peng Z Y. Quantitative analysis of the control of physiological phenomena by plant hormones. Plant Physioogyl,1995,95: 486-494.
[58]Swain S M, Olszewski N E. Genetic analysis of gibberellin signal transduction[J]. Plant Physioogyl,1996,112:11-17.
[59]Frohman M A, Dush M K, Mrtin G R. Rapid production of full- length cDNAs from rare transcripts:amplification using single gene-specific oligonuleotide primer [J]. Proc. Natl. Acad. Sci. USA,1988,85(23):8998-9002.
[60]Murayuma I N, Rakow T L and Muruyama H I. cRACE:A simple method for identfication of the 5'ends of mRNAs [J]. Nucleic Acids Res.,1995,23:3796-3797.
[61]Diachenko L B, Ledesm A J, Chenchik A A, Siebert P D. Combing the techniques of RNA finger printing and differential display to obtain differentially expressed mRNA [J]. Biochem Biophys. Res. Commol/Lun.,1996,219:824-829.
[62]雷娟利,徐志豪. 转座子在植物功能基因组学中的应用[J].浙江农业学报,2002,14(5)：291-296.
[63]杨玲玲.RACE法克隆紫花苜蓿光敏色素A基因[D].郑州：河南农业大学硕士学位论文,2008年6月.
[64]王少丽,盛承发,乔传令.cDNA末端快速扩增技术及其应用[J]. 遗传,2004,26(3)：419-423.
[65]Schaefer B C. Resolution in rapid amplification of cDNA ends:New strategies for polymerase chain reaction cloning of full-length Cdna ends [J]. Anal. Biochem.,1995,227:255-273.
[66]钟涛.cDNA末端快速扩增技术新进展[J].国外医学分子生物学分册,2002,24(1)：7-11.
[67]郝敏,谷守芹,韩建民,董金皋.cDNA末端扩增技术的研究进展[J].河北林果研究,2006,21(2)：157-161.
[68]邱为民,张思仲,武辉,张戈,肖翠英.一种新的cDNA末端快速扩增获取全长cDNA的方法[J].遗传,2001,23(5)：480-482.
[69]Cheng F, Brown S K,Weeden N F.DNA extraction protocol from various tissues in woody species[J]. HortScience,1997,2(5):921-922.
[70]谭晓风,曾艳玲,乌云塔娜,等.雪花梨及其亲缘品种S基因型的确定[J].果树学报,2006,23(3)：355-358.
[71]Chang S,Puryear J.Caimey J.A simple and effecient method for isolating RNA from pine trees[J].Plant Mol Biol Reptr.1993:11:113-116.
[72]王壮伟,渠慎春,章镇,张君毅.苹果属RNA高效快速提取新方法[J]. 果树学报2004,21(4)：385-387.
[73]李晓芳,李茂福,韩振海,等.‘鸭梨’芽变‘闫庄梨’自交亲和性分子机制初 步研究[J].园艺学报,2008,35(1)：13-18.
[74]张妤艳,吴俊,衡伟,张绍铃,,等.京白梨等品种S基因型鉴定及新基因S28和S30的核苷酸序列分析[J].园艺学报,2006,33(3)：496-500。
[75]谭晓风,乌云塔娜,李秀根,等.中国梨品种自交不亲和新基因的分离鉴定[J].中南林学院学报,2005,25(1)：1-3.
[76]乌云塔娜.中国白梨自交不亲和基因的分离鉴定[D].长沙：中南林学院,2003年6月.
[77]张妤艳,张绍铃,吴俊,张瑞萍,李秀根.八月酥等14个梨品种自交不亲和基因(S基因)型的鉴定[J].果树学报,2007,24(2)：135-139.
[78]张春芳,李茂福,韩振海,龙慎山,李天忠.秋子梨等九个品种S基因型的鉴定[J].北方园艺,2008(11)：：131-133.
[79]杨谷良,谭晓风.梨自交不亲和新基因S35-Rnase的克隆与表达分析[J].园艺学报,2007,34(3)：751-754.
[80]衡伟,张绍铃,张好艳,吴俊,李秀根.12个梨品种S基因型的鉴定[J].园艺学报2007,34(4)：853-858.
[81]Malnoy M, Reynoird J P, Mourgues F, et al. A method for isolating total RNA from pear leaves[J].Plant Mol Biol Reptr,2001,19:69-74.
[82]孙长悦.梨组织RNA提取方法研究[J].北方园艺2009(3)：19-21.
[83]赵巍巍,宗成文,曹后男,陈蕾,朴日子.葡萄花序总RNA提取方法研究[J].安徽农业科学,2009,37(32)：16161-1616.
[84]刘晓菊,洪海波,李敏,刘媛,杨克强.改良CTAB法提取核桃总RNA试验[J].山东农业科学2008,1：97-99.
[2]毕晓颖,吴禄平,安利佳.一个与苹果属显性矮生主基因Dw连锁的RAPD标记[J].园艺学报2002,29(1)：1-4.
[3]张所兵.水稻半矮杆突变体株高遗传分析及eui基因定位[D].南京：南京农业大学,2006年6月.
[4]张达,王军虹,王豫颖,等.矮化大豆突变体GA3调控[J].大豆科学,2008,37(3)：456-460.
[5]朴永虎.延边小香水梨(Pyrus. ussuriensis Maxim)偶然实生矮化突变体的评价研究[D]. 延吉：延边大学硕士论文,2010年6月.
[6]孟金陵.植物生殖遗传学[M].北京：科学出版社,1995.
[7]Lewis D.Incompatibility in floiwng plants[J]. Recept Recognition Ser,1976,A2:167-198.
[8]张绍铃,平伸,徐国华,等.梨自交不亲和及其突变品种花柱内S4和S4SM基因的表达与作用比较[J].植物学报,2001,43(11)：1172-1178.
[9]辜青青.利用PCR-RFLP技术鉴定部分沙梨(Pyrus pyrifolia Nakai)品种S基因型[D].武汉：华中农业大学硕士论文,2006,7-8.
[10]Brewbaker J L.Polen Cytology and Self-incompatibility Systems in Plants[J].Hered,1957,48:271-277.
[11]Vaughan S P,Russel L K,Sargent D J, et al.Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype[J].Theor Appl Genet 2006,112:856-866.
[12]Sylvain B,Vincent C,Xavier V.A general model to explore complex dominance patterns in plant sporophytic self-incompatibility systems[J].Genetics,2007.175:1351-1369.
[13]王茂广.孢子体型自交不亲和反应臂重复蛋白ARC1[J].生命科学,2007,19(1)：86-90.
[14]Kikuchi A. Investigation in 1927 and 1928, Pater clinical incompatibility in the Japanese pear [J].Okitse.hort.Soc.1929,24:1-6.
[15]Ushikoshi M, Tokuyasu K. Studies on pollination and fertilization in pear [J]. Okitse. Hort. Soc,1930,25:135-152.
[16]张绍铃,曹生民,吴华清.果树自交不亲和性基因型及其鉴定方法[J].果树学报,2003,20(05)：358-363.
[17]曾艳玲.鹅梨S基因型的鉴定及S基因的cDNA克隆[D].长沙：中南林业科技大学,2006.
[18]乌云塔娜,谭晓风,曹玉芬,等.白梨品种4个新S基因的分离与鉴定[J].园艺学报,2007,34(04)：859-864.
[19]乌云塔娜,谭晓风,毕方铖,等.中国白梨S基因研究Ⅰ：7个品种S基因型的确定和2个新S基因的鉴定[J].中南林学院学报,2005,25(04)：7-12.
[20]谭晓风,毕方铖,乌云塔娜.中国白梨S基因研究Ⅱ：9个主栽品种S基因型的确定及S29-RNase新基因的分离鉴定[J].中南林学院学报,2005,25(04)： 13-16.
[21]袁德义,谭晓风,张琳,等.新高系梨10个品种S基因型的鉴定[J].园艺学报,2007,34(6)：1353-1360.
[22]Ishimizu T, Inoue K, Shimonaka M, et al. PCR-based method for identifying the S-genotypes of Japanese pear cultivars [J].Theor Appl Genet,1999,98:961-967
[23]张春芳,李茂福,韩振海,等.秋子梨等九个品种S基因型的鉴定[J].北方园艺,2008(11)：131—134.
[24]衡伟,张绍铃,张妤艳,等.12个梨品种S基因型的鉴定[J].园艺学报,2007,34(04)：853-858.
[25]Nrioka N,Norilka S,Ohnishi Y. Molecular cloning and nucleotide sequenece of cDNA encoding S-allele specific stylar RNases in a self-incompatible cultivar and its self-compatible mutant of Japanese pear[J]. J. Biochem.,1996, (120):335-345.
[26][美]谢纳.生物芯片分析[M].北京：科学出版社,2004,86-227.
[27]虞慧芳,曹家树,王永勤.植物矮化突变体的激素调控[J].生命科学,2002,14(2)：85-88.
[28]Fu X,Richards DE,Air-ali T et al,Gibberellin-mediated proteasome-dependent degradation of the barley DELLAprotein [J].SLNI repressorPlant Cell,2002,14:3191-3200.
[29]Frohman M A, Dush M K, Mrtin G R. Rapid production of full- length cDNAs from rare transcripts: amplification using single gene -specific oligonuleotide primer[J]. Proc. Natl. Acad. Sci. USA,1988,85(23):8998-9002.
[30]杨铁钊,杨欣玲,殷全玉,等.烟草株高变异体的茎尖内源激素含量变化及其对外源激素的响应[J].植物生理学通讯,2006,42(4)：643-647.
[31]Hedden P, Phillips A L. Gibberellin metabolism: new insights revealed by the genes[J]. Trends in Ptant Science,2000,5(12):523-530.
[32]Ohara O, Dorit R L, Gillbert W. One-sided polymerase chain reaction: the amplification of Cdna[J]. Proc. Natl. Acad. Sci. USA,1989,86(15):5673-5677.
[33]Loh E Y, Elliot J F, Cwirla S, Lanier L L,Davis M M. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain[J]. Science, 1989,243 (4888):217-220.
[34]Schaefer B C. Resolution in rapid amplification of cDNA ends: New strategies for polymerase chain reaction cloning of full-length C dna ends[J]. Anal. Biochem.,1995,227:255-273.
[35]王月华,韩烈保,曾会明,等.植物赤霉素矮化突变体研究进展术[J].中国生物工程杂志,2006,26(8)：22-27.
[36]伍涛,曹家树,虞慧芳.赤霉素合成基因的克隆以及其相关矮化突变体[J].细胞生物学杂志,2005,27：157-164.
[37]Dill A, Jung HS, Sun TP. The DELLA motif is essential for gibberellin-induced degradation of RGA[J]. Proceedings of the National Academy of Sciences,2001, 98:14162-14167.
[38]Pysh L D,Wysocka-Diller J W,Camilleri C,Bouchez, et al.The GRAS family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes [J].Plant J.18,111-119.
[39]Peng J,Carol P,Richards DE,et al.The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses[J].GenesDev,1997,11:3194-8205.
[40]Peng J,Richards D E,Hartley N M, et al.'Green revolution'genes encode mutant gibberellin response modulators[J].Nature,1999,400,256-261.
[41]Chandler PM,Poll AM,Ellis M et al.Mutants at the Sienderl locus of barley Himalaya[J].Molecular and physiological characterization.PlutPhysiol,2002,129:181-190.
[42]Boss P K,Thomas M R. Association of dwarfism and floral in duction with a grape 'green revolution'mutation[J].Nature,2000.1416:847-850.
[43]Ikeda A,Ueguchi-Tanaka M,Sonoda Y,et al.Slender rice,aconstimtive gibberellin response mutant,is caused by a null mutation of the SLRl gene.an ortholog of the height regulating gene,GAI/RGA/RHT/D8[J].Plant Cell,2001.13:999-1010.
[44]Lee S,Cheng H,Peng J, et al.Gibberelin regulates Arabidopsis seed germination via RGL2,a GAI/RGA-like gene whose expression is up-regulated folowing imbibition[J]. Genes Dev,2002,16:646-658.
[45]Ogawa M,Kusano T,Sano H et al.Rice gibberellin-insensitive gene homolog,OsGAI, encodes a nuclear localized protein Capable of gene activation at transcriptional level [J].Gene,2002,45:21-29.
[46]Itoh H,Uegechi-Tanaka M,Sato Y,et al. The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE 1 in nuclei[J]. Plant Cell,2002,14:57-70.
[47]黄先忠,马正强.DELLA家族蛋白与植物生长发育的关系[J].植物生理学通讯,2004.40(5)：529-532.
[48]Salchert K, Bhalerao R, Koncz-Kalman Z, Koncz C. Control of cell elongation and stress responses by steroid hormones and carbon catabolic repression in plants[J]. Series B, Biological Sciences,1998,353(1374):1517-1520.
[49]Chory J, Nagpal P, Peto C A. Phenotypic and gnetic analysis of det2, a new mutant that affects light-regulated seedling development in Arabidopsis[J]. The Plant Cell,1991,3:445-459.
[50]Kauschmann A,Jessop A,Koncz C,et al. Genetic evidence for an essential role of brassiriosteroids in plant development[J].Plant J,1996.9:701-713.
[51]Friedrichaen D M, Joazeiro, Hunter T,Chory J. Brassinosteroid-insensitive-I is a ubiquitously excpressed leucine-rich repeat roceptor serine/threonine kinase[J]. Plant Physiology,2000,123:1247-1255.
[52]Yang T, Davies PJ, Reid JB. Genetic dissection of the relative roles of auxin and gibberellin in the regulation of stem elongation in intact light-grnwn peas[J]. Plant Physiology,1996,110:1029-1034.
[53]Sato Y, Sentoku N, Miura Y, Hirochika H, Kitano H, Matsuoka M. Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants[J]. The European Molecular Biology Organization Journal,1999,18(4):992-1002.
[54]Kusaba S, Fukumoto M, Honda C, Yamaguchi I, Sakamoto T, Kano M Y Decreased GAi content caused bythe overoxprossion of OSH1 is acoompanied by suppression of GA20-oxidaae geneexpression[J].Plant Physiology,1998.117:1179-1184.
[55]Tsiantis M, Brown M I N, Skibinski G.. Disruption of auxin transport is associated with aberrant leaf development in maize[J]. Plant Physiologx 1999, 121(4):1163-1168.
[56]Reiter W D, Chapple C C S, Somerville C R. Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis[J]. Science,1993,261:1032-1035.
[57]Weyers J DB, Paterson N W, Peng Z Y. Quantitative analysis of the control of physiological phenomena by plant hormones. Plant Physioogyl,1995,95: 486-494.
[58]Swain S M, Olszewski N E. Genetic analysis of gibberellin signal transduction[J]. Plant Physioogyl,1996,112:11-17.
[59]Frohman M A, Dush M K, Mrtin G R. Rapid production of full- length cDNAs from rare transcripts:amplification using single gene-specific oligonuleotide primer [J]. Proc. Natl. Acad. Sci. USA,1988,85(23):8998-9002.
[60]Murayuma I N, Rakow T L and Muruyama H I. cRACE:A simple method for identfication of the 5'ends of mRNAs [J]. Nucleic Acids Res.,1995,23:3796-3797.
[61]Diachenko L B, Ledesm A J, Chenchik A A, Siebert P D. Combing the techniques of RNA finger printing and differential display to obtain differentially expressed mRNA [J]. Biochem Biophys. Res. Commol/Lun.,1996,219:824-829.
[62]雷娟利,徐志豪. 转座子在植物功能基因组学中的应用[J].浙江农业学报,2002,14(5)：291-296.
[63]杨玲玲.RACE法克隆紫花苜蓿光敏色素A基因[D].郑州：河南农业大学硕士学位论文,2008年6月.
[64]王少丽,盛承发,乔传令.cDNA末端快速扩增技术及其应用[J]. 遗传,2004,26(3)：419-423.
[65]Schaefer B C. Resolution in rapid amplification of cDNA ends:New strategies for polymerase chain reaction cloning of full-length Cdna ends [J]. Anal. Biochem.,1995,227:255-273.
[66]钟涛.cDNA末端快速扩增技术新进展[J].国外医学分子生物学分册,2002,24(1)：7-11.
[67]郝敏,谷守芹,韩建民,董金皋.cDNA末端扩增技术的研究进展[J].河北林果研究,2006,21(2)：157-161.
[68]邱为民,张思仲,武辉,张戈,肖翠英.一种新的cDNA末端快速扩增获取全长cDNA的方法[J].遗传,2001,23(5)：480-482.
[69]Cheng F, Brown S K,Weeden N F.DNA extraction protocol from various tissues in woody species[J]. HortScience,1997,2(5):921-922.
[70]谭晓风,曾艳玲,乌云塔娜,等.雪花梨及其亲缘品种S基因型的确定[J].果树学报,2006,23(3)：355-358.
[71]Chang S,Puryear J.Caimey J.A simple and effecient method for isolating RNA from pine trees[J].Plant Mol Biol Reptr.1993:11:113-116.
[72]王壮伟,渠慎春,章镇,张君毅.苹果属RNA高效快速提取新方法[J]. 果树学报2004,21(4)：385-387.
[73]李晓芳,李茂福,韩振海,等.‘鸭梨’芽变‘闫庄梨’自交亲和性分子机制初 步研究[J].园艺学报,2008,35(1)：13-18.
[74]张妤艳,吴俊,衡伟,张绍铃,,等.京白梨等品种S基因型鉴定及新基因S28和S30的核苷酸序列分析[J].园艺学报,2006,33(3)：496-500。
[75]谭晓风,乌云塔娜,李秀根,等.中国梨品种自交不亲和新基因的分离鉴定[J].中南林学院学报,2005,25(1)：1-3.
[76]乌云塔娜.中国白梨自交不亲和基因的分离鉴定[D].长沙：中南林学院,2003年6月.
[77]张妤艳,张绍铃,吴俊,张瑞萍,李秀根.八月酥等14个梨品种自交不亲和基因(S基因)型的鉴定[J].果树学报,2007,24(2)：135-139.
[78]张春芳,李茂福,韩振海,龙慎山,李天忠.秋子梨等九个品种S基因型的鉴定[J].北方园艺,2008(11)：：131-133.
[79]杨谷良,谭晓风.梨自交不亲和新基因S35-Rnase的克隆与表达分析[J].园艺学报,2007,34(3)：751-754.
[80]衡伟,张绍铃,张好艳,吴俊,李秀根.12个梨品种S基因型的鉴定[J].园艺学报2007,34(4)：853-858.
[81]Malnoy M, Reynoird J P, Mourgues F, et al. A method for isolating total RNA from pear leaves[J].Plant Mol Biol Reptr,2001,19:69-74.
[82]孙长悦.梨组织RNA提取方法研究[J].北方园艺2009(3)：19-21.
[83]赵巍巍,宗成文,曹后男,陈蕾,朴日子.葡萄花序总RNA提取方法研究[J].安徽农业科学,2009,37(32)：16161-1616.
[84]刘晓菊,洪海波,李敏,刘媛,杨克强.改良CTAB法提取核桃总RNA试验[J].山东农业科学2008,1：97-99.