蒙古沙冬青干旱诱导表达SMART cDNA文库的构建及序列分析
|
摘要
沙冬青是我国西北荒漠区特有的唯一常绿旱生阔叶灌木,具有很强的抗寒和抗旱等抗逆特性。目前对于沙冬青抗逆性分子机理和抗逆基因的分离鉴定集中于抗寒性方面,有关其抗旱基因的分离和鉴定尚未见报道。本论文以蒙古沙冬青(Ammopiptanthus mongolicus)为材料,采用SMART(Switching Mechanism At 5’end of RNA Transcript method)技术构建了其在干旱胁迫下的全长cDNA文库,并对文库中的部分克隆进行了5’端测序和序列分析,为进一步开展表达谱分析和重要抗旱基因的克隆鉴定工作奠定了基础。
实验首先对经典热酚法加以改进,解决了蒙古沙冬青叶片总RNA提取中多糖和多酚类物质污染严重的问题,获得符合建库要求的高质量总RNA。此外,还发现Promega公司的PolyATtract? mRNA Isolation Systems更适合于沙冬青mRNA的分离纯化。
以不同干旱胁迫处理的蒙古沙冬青混合mRNA为模板合成cDNA,构建了SMART全长cDNA文库。原始文库的滴度和重组率分别为1.88×107 pfu/mL和97.87%,扩增文库的滴度为4.32×1010pfu/mL;插入片段大小在500~2500 bp之间,表明所建文库质量较高。
将所构建的λTriplEx2文库转化成pTriplEx2质粒文库,经菌液PCR鉴定获得3500个阳性克隆。将其中960个克隆进行5’端测序,共获得875个有效EST序列。将EST序列去载体后进行拼接,获得97个Contig和509个Singlet,总计606个Unigene。这些Unigene通过功能注释可分成19个大类,其中与蛋白质合成、修饰、转运和降解相关的基因所占比例最高,其次是一些具有普通功能的基因和参与小分子及离子运输与代谢的基因,与能量代谢、基因转录、细胞壁和膜的形成、信号转导、细胞周期、胞内运输和RNA加工与修饰等过程相关的基因也占一定比例。有2.65%的Unigene未得到功能注释。
As an only evergreen broad-leaf shrub in the northwest desert of China, Ammopiptanthus mongolicus shows very strong resistance to both drought and cold stresses. At present, studies on the sterss-resistant molecular mechanisms and the involved genes in the plant are limited to its cold resistance only, and report about the isolation and characterization of the drought-resistant genes from the plant has not been found as yet. In the present study, a drought-induced full-length cDNA library of A. mongolicus was constructed by using SMART (switching mechanism at 5’-end of RNA transcript) cDNA library construction method. In addition, 5’-sequencing and the seqnence analyses of some clones from the library were carried out. The work laid a foundation for both the expression profile analysis and the cloning and characterization of drought-resistant genes from the plant in the future.
Firstly, the classical hot phenol method was improved and the contamination problems of total RNA by polyphenols and polysaccharides during its extraction from A. mongolicus were solved, and as a result, high qulity total RNA meeted the requirements for SMART cDNA library construction were extracted from the plant. What’s more, the PolyATtract? mRNA Isolation Systems produced by Promega company was proved more suitable for the mRNA isolation from total RNA of A. mongolicus.
Subsequently, using mixed mRNA isolated from different A. mongolicus samples treated under different drought conditions as template for cDNA synthesis, we constructed a full-length cDNA library with SMART cDNA library construction method. The titer of the unamplified library is 1.88×107; the recombination percentage of the library reaches up to 97.87%; the titer of the amplified library is 4.32×1010 pfu/ml; the sizes of the insert cDNA fragments ligated toλTriplEx2 vector ranges from 0.5 to 2.5kb. All the results suggest that the library has a higher qulity.
Furthermore, part of the unamplifiedλTriplEx2 library was converted to pTriplEx2 library, and 3500 positive clones were selected from the plasmid library by PCR. Up to date, 960 positive clones have been sequenced from their 5’-end, and 875 useful EST sequences were obtained. After removing the vetor sequences and clustering them, we finally obtained 97 contig and 509 singlet, summing into 606 Unigene. All these Unigene can be classified into 19 clusters. Of which, the genes related to synthesis, modification, transport and degradation of proteins are the most, and the genes with genaral functions and the genes associated with the transport or metabolism of small molleculars and irons rank second. The genes associated with energy metabolism, transcription, cell wall and membrane formation, signal transduction, cell cycle, intracellular transportation, RNA processing and modification, and so on, respectively are also involved in the Unigene. Finally, there exist 2.65 percent Unigene which have not been functionally annotated as yet.
实验首先对经典热酚法加以改进,解决了蒙古沙冬青叶片总RNA提取中多糖和多酚类物质污染严重的问题,获得符合建库要求的高质量总RNA。此外,还发现Promega公司的PolyATtract? mRNA Isolation Systems更适合于沙冬青mRNA的分离纯化。
以不同干旱胁迫处理的蒙古沙冬青混合mRNA为模板合成cDNA,构建了SMART全长cDNA文库。原始文库的滴度和重组率分别为1.88×107 pfu/mL和97.87%,扩增文库的滴度为4.32×1010pfu/mL;插入片段大小在500~2500 bp之间,表明所建文库质量较高。
将所构建的λTriplEx2文库转化成pTriplEx2质粒文库,经菌液PCR鉴定获得3500个阳性克隆。将其中960个克隆进行5’端测序,共获得875个有效EST序列。将EST序列去载体后进行拼接,获得97个Contig和509个Singlet,总计606个Unigene。这些Unigene通过功能注释可分成19个大类,其中与蛋白质合成、修饰、转运和降解相关的基因所占比例最高,其次是一些具有普通功能的基因和参与小分子及离子运输与代谢的基因,与能量代谢、基因转录、细胞壁和膜的形成、信号转导、细胞周期、胞内运输和RNA加工与修饰等过程相关的基因也占一定比例。有2.65%的Unigene未得到功能注释。
As an only evergreen broad-leaf shrub in the northwest desert of China, Ammopiptanthus mongolicus shows very strong resistance to both drought and cold stresses. At present, studies on the sterss-resistant molecular mechanisms and the involved genes in the plant are limited to its cold resistance only, and report about the isolation and characterization of the drought-resistant genes from the plant has not been found as yet. In the present study, a drought-induced full-length cDNA library of A. mongolicus was constructed by using SMART (switching mechanism at 5’-end of RNA transcript) cDNA library construction method. In addition, 5’-sequencing and the seqnence analyses of some clones from the library were carried out. The work laid a foundation for both the expression profile analysis and the cloning and characterization of drought-resistant genes from the plant in the future.
Firstly, the classical hot phenol method was improved and the contamination problems of total RNA by polyphenols and polysaccharides during its extraction from A. mongolicus were solved, and as a result, high qulity total RNA meeted the requirements for SMART cDNA library construction were extracted from the plant. What’s more, the PolyATtract? mRNA Isolation Systems produced by Promega company was proved more suitable for the mRNA isolation from total RNA of A. mongolicus.
Subsequently, using mixed mRNA isolated from different A. mongolicus samples treated under different drought conditions as template for cDNA synthesis, we constructed a full-length cDNA library with SMART cDNA library construction method. The titer of the unamplified library is 1.88×107; the recombination percentage of the library reaches up to 97.87%; the titer of the amplified library is 4.32×1010 pfu/ml; the sizes of the insert cDNA fragments ligated toλTriplEx2 vector ranges from 0.5 to 2.5kb. All the results suggest that the library has a higher qulity.
Furthermore, part of the unamplifiedλTriplEx2 library was converted to pTriplEx2 library, and 3500 positive clones were selected from the plasmid library by PCR. Up to date, 960 positive clones have been sequenced from their 5’-end, and 875 useful EST sequences were obtained. After removing the vetor sequences and clustering them, we finally obtained 97 contig and 509 singlet, summing into 606 Unigene. All these Unigene can be classified into 19 clusters. Of which, the genes related to synthesis, modification, transport and degradation of proteins are the most, and the genes with genaral functions and the genes associated with the transport or metabolism of small molleculars and irons rank second. The genes associated with energy metabolism, transcription, cell wall and membrane formation, signal transduction, cell cycle, intracellular transportation, RNA processing and modification, and so on, respectively are also involved in the Unigene. Finally, there exist 2.65 percent Unigene which have not been functionally annotated as yet.
引文
1张艳敏.植物逆境应答的分子机制及转基因研究[J].河北农业科学,2003,7(4):33-37
2 Levitt J.Response of Pinata to environmental stresses.Water,radiation,salt and other stresses[M].New York:Aeademic Press,1980,325-358
3 Tumen NC.Drought resistance and adaptation to water deficits in crop paints.In:Harry Muss all stress physiology in crop plants[M].New York:John Wiley and Sons,1979,343-372
4户连荣,郎南军,郑科.植物抗旱性研究进展及发展趋势[J].安徽农业科学,2008,36(7):2652-2654
5贾晋平.玉米全长cDNA文库的构建及生物信息学分析[D],中国农业大学博士论文,2006
6 Yancy PH,Clark ME,Hand SC,etal.Living with water stress: evolution of osmolyte systems[J].Science,1982,217:1214-1222
7 Delauney AY , Verma DPS.Proline biosynthesis and osmoregulation in plants[J].Plant J,1993,4:215-223
8 Rhodes D,Handa S,Bressan RA.Metabolic changes associated with adaption of plant cells to water stress[J].Plant Physiol,1986,82:890-903
9 Csonka LN.Physiological and genetic responses of bacterial to osmotic stress[J].Microbiol Rev.1989,53:121-147
10 Whatmore AM,Chudek JA,Reed RH.The effects of osmotic upshock on the extracellular solute pools of Bacillus subtilis[J].J Gen.Microbiol, 1990,136:2527-2535
11 Zhu JK , Hasegawa PM , Bressan RA.Molecular aspects of osmotic stress in plants[J].Crit Rev Plant Sci,1997,16:253-277
12 Asada K.Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants[J].Physiol Plant,1992,85:235-241
13 Ball L , Accotto GP , Bechtold U , etal.Evidence for a Direct Link between GlutathioneBiosynthesis and Stress Defense Gene Expression in Arabidopsis[J].Plant Cell,2004,16:2448-2462
14 Romero C,Belles JM,Vaya JL,etal.Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants:pleiotropic phenotypes include drought tolerance[J].Planta, 1997,201:293-297
15 Yamaguchi-Shinozaki K , Koizumi M , Urao S , etal.Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein[J].Plant Cell Physiol,1992,33:217-224
16 Albert HA,Martin T and Sun SSM.Structure and expression of a sugarcane geneencoding a housekeeping phosphoenolpyruvate carboxylase[J].Plant Mol Biol,1992,20:663-671
17 Adam E,Szell M,Szekeres M,etal.The developmental and tissue-specific expression of tobacco phytochrome-A genes[J].Plant J,1994,6:283-293
18 Tonoike H,Han IS,Jongewaard I,etal.Hytocotyl expression and light down regulation of the soybean tubulin gene,tubB1[J].Plant J,1994,5:343-351
19 Abe H,Yamaguchi-Shinozaki K,Urao T,etal.Role of Arabidopsis MYC and MYB homologs in drought-and abscisic acid-regulated gene expression[J].Plant Cell,1997,9:1859-1868
20 Uno Y,Furihata T,Abe H,etal.Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions[J]. Proc. Natl.Acad.Sci.USA , 2000 ,97:11632-11637
21 Liu Q,Kasuga M,Sakuma Y,etal.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low-temperature-responsive gene expression ,respectively,in Arabidopsis[J].Plant Cell. 1998,10:1391-1406
22 Kasuga M,Liu Q,Miura S,etal.Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J].Nat Biotechnol,1999,17 (3):287-291
23 Lee JT, Prasad V, Yang PT,etal.Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield[J].Plant Cell &Environment,2003,26:1181-1190
24 Trewavas AJ,Malho R.Signal Perception and transduction:the origin of the phnotype[J].Plant Cell,1997,9:1181-1195
25 Shinozaki K,Yamaguchi-Shinozaki K,Mizoguchi T,etal.Role of MAP kinase,two component histidine kinase and calcium dependent protein kinase in water stress response.Gadal P,etal.(eds.)Protein Phosphorylation in Plants[J].Institute de Biotechnologie des Plantes Orsay,France,1998,11-17
26 Schroeder JI,Allen GJ,Hugouvieux V,Kwak JM,Waner D.Guard cell signal transduction[J].Annu Rev Plant Mol Boil,2001,52:627-658
27 Kwak JM,Mori IC,Pei ZM,Leonhardt N,Torres MA,Dangl JL,Bloom RE,Bodde S,Jones JD,Schroeder JI.NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis[J].EMBO J,2003,22:2623-2633
28 Xiong L,Schumaker KS,Zhu JK.Cell signaling during cold,drought,and saltstress[J].Plant Cell,2002,14: S165-S183
29 Kim KN,Cheong YH,Grant JJ,Pandey GK,Luan S.CIPK3,a Calcium Sensor Associated Protein Kinase That Regulates Abscisic Acid and Cold Signal Transduction in Arabidopsis[J]. Plant Cell,2003,15: 411-423
30 Zhu J,Gong Z,Zhang C,Song CP,Damsz B,Inan G,Koiwa H,Zhu JK,Hasegawa PM,Bressan RA.OSM1/SYP61: a syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated responses to abiotic stress[J].Plant Cell,2002,14: 3009-3028
31 Xiong L,Lee H,Ishitani M,Zhu JK.Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus in Arabidopsis[J].J Biol Chem,2002,277: 8588-8596
32 Shinozaki K,Yamaguchi-Shinozaki K,Seki M.Regulatory network of gene expression in the drought and cold stress responses[J].Curr Opin Plant Biol,2003,6:410-417
33 Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression , respectively , in Arabidopsis[J].Plant Cell,1998,10:1391-1406
34 Ishitani M,Xiong L,Stevenson B,Zhu JK.Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis:interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways[J].Plant Cell,1997,9:1935-1949
35 GmblerΜ,Hoffman BJ.A simple and very elflent method for generating cDNA library[J].Gene,1983,25:263-269
36许兰珍,何永睿,姜国金,刘小丰,陈善春.cDNA文库构建及其在植物抗性研究中的应用[J].安徽农业科学,2007,35(3):660-662,664
37 Carninci P,Shibata Y,Hayatsu N,Sugahara Y,Shibata K,Itoh M,Konno H,Okazaki Y , Muramatsu M , Hayashizaki Y.Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
38 Kato S,Sekine S,Oh SW,Kim NS,Μmezawa Y,Abe N,Yokoyama-Kobayashi M,Aoki T.Construction of a human full-length cDNA bank[J].Gene,1994,150(2):243-250
39 Seki M,Carninci P,Nishiyama Y,Hayashizaki Y and Shinozaki K.High-efficiency cloning Arabidopsis full-length cDNA by biotinylated CAP trapper[J].The plant Journal,1998,15(5):707-720
40 Suzuki Y,Sugano S.Construction of a full-length enriched and a 5’-end enriched cDNA library using the oligo-capping method[J].Methods Mol Biol,2003,221 (1):73-91
41 Suzuki Y,Yoshitomo-Nakagawa K,Maruyama K,Suyama A,Sugano S.Construction and characterization of a full-length-enriched and a 5’-end-enriched cDNA library[J].Gene,1997,200(122):149-156
42 Zhu YY,Machleder EM,Chenchik A,Li R,Siebert PD.Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction[J].Biotechniques,2001,30(4):892-897
43 Efimov VA,Chakhmakhcheva OG,Archdeacon J,Fernandez J,Fedorkin ON,Dorokhov Yu L,Atabekov JG.Detection of the 5’-cap structure of messenger RNAs with the use of the cap-jumping approach[J].Nucl Acids Res,2001,29(22):4751-4759
44 Carninci P,Shibata Y,Hayatsu N,Sugahara Y,Shibata K,ItohM,Konno H,Okazaki Y , Muramat su M , Hayashizaki Y.Nor-malization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
45 Carninci P,Kvam C,Kitamura A,Ohsumi T,Okazaki Y,Itoh M,Kamiya M,Shibata K,Sasaki N,Izawa M,Muramatsu M,Hayashizaki Y,Schneider C.High-efficiency full-length cDNA cloning by biotinylated CAP trapper[J].Genomics,1996,37 (3):327-336
46 Carninci P,Westover A,Nishiyama Y,Ohsumi T,Itoh M,Nagaoka S,Sasaki N,Okazaki Y,Muramat su M,Schneider C,Hayashizaki Y.High efficiency selection of full-length cDNA by improved biotinylated captrapper[J].DNA Res,1997,4(1): 61-66
47 Edery I,Chull,Sonenberg N,Pelletier J.An efficient strategy to isolate full-length cDNAs.based on an mRNA cap retention procedure(CAPture)[J].Mol Cell Biol,1995,15(6):3363-3371
48 Sugahara Y , Carninci P , Itoh M , etal.Comparative evaluation of 5’-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries[J].Gene,2001,263:93-102
49 Maruyama K,Sugano S.Oligo-capping:a simple method to replace the cap structure of eukaryotic mRNAs with oligoriboucleotides[J].Gene,1994,138(1-2):171-174
50 Suzuki Y,Yoshitomo-Nakagawa K,Maruyama K,Suyama A, Sugano S. Construction and characterization of a full length-enriched and a 5’-end-enriched cDNA library[J].Gene,1997,200(1-2):149-156
51 Suzuki Y,Sugano S.Construction of full-length-enriched cDNA libraries.The oligo-capping method[J].Methods Mol Biol,2001,175:143-153
52 Suzuki Y,Sugano S.Construction of a full-length enriched and a 5’-end enriched cDNA library using the oligo-capping method[J].Methods Mol Biol,2003,221:73-91
53 Chenchik A,Moqadam F.Siebert P.RNA:Isolation,Analysis and Synthesis[M].New York:Wiley-Liss,l996,273-32l
54毛新国,景蕊莲,孔秀英,赵光耀,贾继增.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7):865-873
55 Zhu YY,Machleder EM,Chenchik A,Li R,Siebert PD.Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction[J]. Biotechniques,2001,30(4):892-897
56 Wellenreuther R,Schupp I,Poustka A,etal.The German cDNA consortium SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones[J].BMC Genomics,2004,36(5):1-8
57董志敏,张宝石,关志霞,常汝镇,邱丽娟.全长cDNA文库的构建方法[J].中国农学通报,2007,22(2):51-53
58王峰,王金发.水稻幼叶及幼穗cDNA文库的构建及初步分析[J].中山大学学报,2005,44(2):74-77
59董海滨,管荣展.双低油菜华双3号幼苗全长cDNA文库的构建[J].南京农业大学学报2005,28(3):123-125
60徐碧玉,苏伟,张建斌,刘菊华,金志强.香蕉果实SMART eDNA文库的构建及利用PCR方法筛选香蕉Actin2基因[J].热带亚热带植物学报2005,13(5):375-380
61蔡勤安,王玉成,张道远,董玉芝.渗透胁迫下白花柽柳cDNA文库的构建及EST分析[J].生物技术,2005,15(3):22-26
62 Carninci P,Nishiyama Y,Westover A,Itoh M,Nagaoka S,Sasaki N, Okazaki Y,Muramatsu M , Hayashizaki Y.Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA[J].Proc Natl Acad Sci USA,1998,95(2):520-524
63 Carninci P,Hayashizaki Y.High-efficiency full-length cDNA cloning[J].Methods Enzymol,1999,303:19-44
64 Carninci P,Shiba a Y,Hayatsu N,Sugahara Y,Shibata K,Itoh M,Konno H,Okazaki Y , Muramatsu M , Hayashizaki Y.Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
65 Carninci P,Waki,Sh ki T,Konno H,Shibata K,Itoh M,Aizawa K,Arakawa T,Ishii Y , Sasaki D , Hayashizaki Y.Targeting a complex transcriptome:the construction of the mouse full-length cDNA encyclopedia[J].Genome Res, 2003,13(6B):1273-1289
66毛新国,孔秀英,赵光耀,贾继增.利用改进的Cap-trapper法构建拟斯卑尔脱山羊草(Ae.speltoides Tausch)全长cDNA文库[J].遗传学报,2005,32 (8):811-817
67 Seki M,Narusaka M,Yamaguchi-Shinozaki K,etal.Arabidopsis encyclopedia using full-length cDNAs and its application.Plant Physiology and Biochemistry[J].2001,39:211-220
68 Seki M, Narusaka M, Ishida J, etal.Monitoring the expression profiles of 7000 Arabidopsis genes under drought,cold,and high-salinity stresses using a full-length cDNA microarray[J]. Plant J, 2002a,31:279-292
69 Seki M,Narusaka M,Kamiya A,et al.Functional annotation of a full-length Arabidopsis cDNA collection[J].Science,2002b,296:141-145
70 Seki M,Ishida J,Narusaka M,etal.Monitoring the expression pattern of around 7000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray[J].Funct.Integr.Genomic,2002c,2:282-291
71 Seki M , Satou M , Sakurai T , et al.RIKEN Arabidopsis full-length cDNA database[J].Trends Plant Sci,2002d,7:562-563
72 Kikuchi S,Satoh K,Nagata T,etal.Collection,mapping,and annotation of over 28000 cDNA clones from japonica Rice[J].Science,2003,301:376-378
73毛新国.小麦族A、S、O二倍体种全长cDNA文库构建及序列初步分析[D].中国农业科学院博士论文,2004
74 Osato N,Itoh M,Konno H etal.A computer-based method of selecting clones for a full-length cDNA project: simultaneous collection of negligibly redundant and variant cDNAs[J].Genome Res,2002,12:1127-1134
75 Kasuga M,Liu Q,Miura S,etal.Improving plant drought,salt,and freezing tolerance by gene transfer of a single Reymond P,Weber H,Damond M, Farmer EE,ifferential gene expression in response to mechanical wounding and insect feeding in Arabidopsis[J].Plant Cell,2000,12:717-719
76 Shinozaki K,and Yamaguchi-Shinozaki K.Gene expressionand signal transduction in water-stress response[J].Plant Physiology,1997,115:327-334
77 Wang R,Guegler K,LaBrie ST,etal.Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate[J].Plant Cell,2000,12:1491-1509
78 Ahn S,Anderson JA,Sorrells ME,etal.Homoeologous relationships of rice,wheatand maize chromosomes[J].Mol.Gen.Genet,1993a,241:483-490
79 Ahn S , and Tanksley SD.Comparative linkage maps of the rice and maize genomes[J].Proc.Natl.Acad.Sci.USA,1993b,90:7980-7984
80 Bennetzen JL,and Ma JX.The genetic colinearity of rice and other cereals on the basis of genomic sequence analysis[J].Curr.Opin.Plant Biol,2003,6:128-133
81 Swigonova Z , Lai J , Ma J , etal.Close split of sorghum and maize genome progenitors[J].Genome Res,2004,14:1916-1923
82李慧卿,马文元,李慧勇.沙冬青抗逆性研究进展[J].世界林业研究,2000,13(5):67-71
83陶玲,李新荣,刘新民,等.中国珍惜濒危荒漠植物保护等级的定量研究[J].林业科学,2001,37(1):52-57
84 Burke MJ,Gusta LV,Quam me HA.Freezing and injury in plants[J].Ann Rev Plant Physiol,1976,27:507-528
85梁秋霞,曹刚强,押辉远.抗冻蛋白在园艺植物遗传改良中的研究进展[J].北方园艺,2006 (6):50-52
86吴建民,幸华,赵志光,等.植物抗冻蛋白的研究进展及其应用[J].冰川冻土,2004,26(4):482-487
87周晓蕾,陈滔滔,王保怀,等.沙冬青抗冻蛋白热滞活性的DSC研究[J].物理化学学报,2001,17(1):66-69
88 Griffith M,Ala P,Yang DSC,etal.Antifreeze protein produced endogenously in winter rye leaves[J].Plant Physiol,1992,100:593–596
89费云标,孙龙华,黄涛,等.沙冬青高活性抗冻蛋白的发现[J].植物学报,1994,36 (8):649-650
90 Huang T,Duman JG.Purification and characterization of thermal hysteresis protein from cold-acclimated kale,Brassica oleracea [J].Cryobiology,1995,32:577-581
91 At1c1 ?,Nalbantolu B.Effect of apoplastic proteins on freezing to lerance in leaves[J].Phytochemistry,1999,50:755-761
92江勇,魏令波,费云标,等.分离和鉴定沙冬青抗冻蛋白质[J].植物学报,1999,41 (9):967-971
93魏令波,江勇,舒念红,等.沙冬青叶片热稳定抗冻蛋白特性分析[J].植物学报,1999,41(8):837-841
94费云标,魏令波,高素琴,等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20):2185-2189
95舒念红,魏令波,高素琴,等.植物抗冻蛋白纯化、活性与二级结构测定[J].中国医学研究与临床,2003,1(12):45-47
96 Wang W,Wei L.Purification of boiling-soluble antifreeze protein from the legumeAmmopiptanthus mongolicus[J].Prep Biochem Biotechnol,2003, 33(1):67-80
97 Wang W,Wei L,Wang G.Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods[J].Chromatogr Sci,2003,41(9):489-493
98王维香.沙冬青抗冻蛋白的分离纯化及特性鉴定[D].大连理工大学博士论文, 2004
99王维香,魏令波,张虎.沙冬青热稳定抗冻蛋白的分离纯化及其部分性质研究[J].四川大学学报(自然科学版),2007,44(4):912-917
100 Fei YB, Cao PX, Gao SQ, etal.Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus[J].Prep Biochem Biotechnol,2008,38(2):172-183
101鲁春芳,尹林克,牟书勇,等.新疆沙冬青叶片氨基酸和蛋白质的测试与分离以及抗冻蛋白的鉴定结果分析[J].武汉植物学研究,2007,25(5):531-534
102 Yun-Biao Fei, Peng-Xiu Cao, Su-Qin Gao,etal.Discovery of a Homolog of Siderophilin in a Plan[J]. Journal of integrative plant biology , 2005 ,47(12):1409-1411
103 Liu M,Shen X,Yin W,etal.Functional analysis of cold-inducible cDNA clones in the legume Ammopiptanthus mongolicus[J].Cryo Letters, 2005,26(4):213-222
104 Liu MQ , Lu CF , Shen X et al.Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus[J].DNA Sequence,2006,17(5):342-349
105刘美芹,沈昕,卢存福,等.一种改进的固相扣除杂交法直接克隆全长差异表达基因[J].北京林业大学学报,2007,29(5):67-72
106陈奕吟.沙冬青低温诱导基因转化烟草及转基因植物的抗寒性研究[D].北京林业大学硕士学位论文,2007
107孙燕.沙冬青CBL1基因的克隆及表达特性研究[D].北京林业大学硕士学位论文,2005
108 Batistic O, Kudla J.Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J].Planta 2004,219: 915-924
109孙芳.沙冬青CBLI基因的功能研究[D].北京林业大学硕士学位论文,2009
110宋健.沙冬青耐寒基因AmGolS的克隆和遗传转化研究[D].山东农业大学硕士学位论文,2007
111郭九峰,孙国琴,沈传进,等.沙冬青cDNA文库的构建和EST分析[J].华北农学报,2007,22(4):37-41
112乔慧蕾,王瑞刚,郭九峰,等.沙冬青脱水素基因的克隆及表达载体的构建[J].华北农学报,2009,(4):89-90
113 Pengxiu Cao,Jian Song,Chunjiang Zhou,etal.Characterization of multiple coldinduced genes from Ammopiptanthus mongolicus and functional analyses of gene AmEBP1[J].Plant Molecule Biology,2008,529-539
114张晗.棉花CBF调控因子在逆境条件下的表达调控及沙冬青CBF基因的克隆.中国农业科学院硕士学位论文,2008
115刘静,王长宪,王斌,等.抗寒基因AmGS高效转化体系的研究[J].山东农业大学学报(自然科学版),2009,40(2):191-194
116 Sambrook,分子克隆实验指南[M].北京:科学出版社,2002,第三版
117李宏,王新力.植物组织RNA提取的难点及对策[J].生物技术通报,1999,1:36-39
118王玉成,杨传平,姜静.木本植物组织总RNA提取的要点与原理[J].东北林业大学学报,2002,30:1-4
119朱昀,王猛,贾志伟,练云,金颖,王国英.一种从富含多糖的玉米幼穗中提取RNA的方法[J].植物学通报,2007,24(5):624-628
120周春娥,段红英,齐力旺.木本植物老根老叶总RNA的提取方法[J].安徽农业科学,2007,35:1831-2832
121高鹏.玉米幼苗淹水诱导表达基因的克隆及其功能分析[M].北京,中国农业大学博士论文,2003
122 Selenge D,Batsuren D,Batriov E Kh,etal. Chemical study of the flora of mongolicus isoflavones of Ammopiptanthus mongolicus[J].Khim Prirodn Soedin.1986,2:242
123田晓明,陈世忠,屠鹏飞,雷连娣.沙冬青地上部分的化学成分研究[J].中国中药杂志.2008,33:10
124 Ainsworth C.Isolation of RNA from floral tissue of Rumex acetosa (sorrel)[J].Plant Mol Biol Rep,1994,12:198-203,212-215
125王昆,王颖,鲍永利,孟祥颖,乌垠,易静文,李玉新.人参叶全长cDNA文库的构建及部分克隆的序列分析[J].分子科学学报,2006,22(1):58-61
126董志敏,李英慧,张宝石,关荣霞,常汝镇,邱丽娟.一种获得大片段克隆的SMART全长cDNA文库构建方法[J].大豆科学,2007,3:223-227
127 Zheng J,Zhao J,Tao Y,Wang J,Liu Y,Fu J,Jin Y,Gao P,Zhang J, Bai Y,Wang G.Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray[J].Plant Mol. Biol,2004,55(6):807-823
128张锦鹏.谷子在干旱逆境中差异表达基因的分离与表达谱分析[D].中国农业大学博士学位论文,2006
2 Levitt J.Response of Pinata to environmental stresses.Water,radiation,salt and other stresses[M].New York:Aeademic Press,1980,325-358
3 Tumen NC.Drought resistance and adaptation to water deficits in crop paints.In:Harry Muss all stress physiology in crop plants[M].New York:John Wiley and Sons,1979,343-372
4户连荣,郎南军,郑科.植物抗旱性研究进展及发展趋势[J].安徽农业科学,2008,36(7):2652-2654
5贾晋平.玉米全长cDNA文库的构建及生物信息学分析[D],中国农业大学博士论文,2006
6 Yancy PH,Clark ME,Hand SC,etal.Living with water stress: evolution of osmolyte systems[J].Science,1982,217:1214-1222
7 Delauney AY , Verma DPS.Proline biosynthesis and osmoregulation in plants[J].Plant J,1993,4:215-223
8 Rhodes D,Handa S,Bressan RA.Metabolic changes associated with adaption of plant cells to water stress[J].Plant Physiol,1986,82:890-903
9 Csonka LN.Physiological and genetic responses of bacterial to osmotic stress[J].Microbiol Rev.1989,53:121-147
10 Whatmore AM,Chudek JA,Reed RH.The effects of osmotic upshock on the extracellular solute pools of Bacillus subtilis[J].J Gen.Microbiol, 1990,136:2527-2535
11 Zhu JK , Hasegawa PM , Bressan RA.Molecular aspects of osmotic stress in plants[J].Crit Rev Plant Sci,1997,16:253-277
12 Asada K.Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants[J].Physiol Plant,1992,85:235-241
13 Ball L , Accotto GP , Bechtold U , etal.Evidence for a Direct Link between GlutathioneBiosynthesis and Stress Defense Gene Expression in Arabidopsis[J].Plant Cell,2004,16:2448-2462
14 Romero C,Belles JM,Vaya JL,etal.Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants:pleiotropic phenotypes include drought tolerance[J].Planta, 1997,201:293-297
15 Yamaguchi-Shinozaki K , Koizumi M , Urao S , etal.Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein[J].Plant Cell Physiol,1992,33:217-224
16 Albert HA,Martin T and Sun SSM.Structure and expression of a sugarcane geneencoding a housekeeping phosphoenolpyruvate carboxylase[J].Plant Mol Biol,1992,20:663-671
17 Adam E,Szell M,Szekeres M,etal.The developmental and tissue-specific expression of tobacco phytochrome-A genes[J].Plant J,1994,6:283-293
18 Tonoike H,Han IS,Jongewaard I,etal.Hytocotyl expression and light down regulation of the soybean tubulin gene,tubB1[J].Plant J,1994,5:343-351
19 Abe H,Yamaguchi-Shinozaki K,Urao T,etal.Role of Arabidopsis MYC and MYB homologs in drought-and abscisic acid-regulated gene expression[J].Plant Cell,1997,9:1859-1868
20 Uno Y,Furihata T,Abe H,etal.Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions[J]. Proc. Natl.Acad.Sci.USA , 2000 ,97:11632-11637
21 Liu Q,Kasuga M,Sakuma Y,etal.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low-temperature-responsive gene expression ,respectively,in Arabidopsis[J].Plant Cell. 1998,10:1391-1406
22 Kasuga M,Liu Q,Miura S,etal.Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J].Nat Biotechnol,1999,17 (3):287-291
23 Lee JT, Prasad V, Yang PT,etal.Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield[J].Plant Cell &Environment,2003,26:1181-1190
24 Trewavas AJ,Malho R.Signal Perception and transduction:the origin of the phnotype[J].Plant Cell,1997,9:1181-1195
25 Shinozaki K,Yamaguchi-Shinozaki K,Mizoguchi T,etal.Role of MAP kinase,two component histidine kinase and calcium dependent protein kinase in water stress response.Gadal P,etal.(eds.)Protein Phosphorylation in Plants[J].Institute de Biotechnologie des Plantes Orsay,France,1998,11-17
26 Schroeder JI,Allen GJ,Hugouvieux V,Kwak JM,Waner D.Guard cell signal transduction[J].Annu Rev Plant Mol Boil,2001,52:627-658
27 Kwak JM,Mori IC,Pei ZM,Leonhardt N,Torres MA,Dangl JL,Bloom RE,Bodde S,Jones JD,Schroeder JI.NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis[J].EMBO J,2003,22:2623-2633
28 Xiong L,Schumaker KS,Zhu JK.Cell signaling during cold,drought,and saltstress[J].Plant Cell,2002,14: S165-S183
29 Kim KN,Cheong YH,Grant JJ,Pandey GK,Luan S.CIPK3,a Calcium Sensor Associated Protein Kinase That Regulates Abscisic Acid and Cold Signal Transduction in Arabidopsis[J]. Plant Cell,2003,15: 411-423
30 Zhu J,Gong Z,Zhang C,Song CP,Damsz B,Inan G,Koiwa H,Zhu JK,Hasegawa PM,Bressan RA.OSM1/SYP61: a syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated responses to abiotic stress[J].Plant Cell,2002,14: 3009-3028
31 Xiong L,Lee H,Ishitani M,Zhu JK.Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus in Arabidopsis[J].J Biol Chem,2002,277: 8588-8596
32 Shinozaki K,Yamaguchi-Shinozaki K,Seki M.Regulatory network of gene expression in the drought and cold stress responses[J].Curr Opin Plant Biol,2003,6:410-417
33 Liu Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression , respectively , in Arabidopsis[J].Plant Cell,1998,10:1391-1406
34 Ishitani M,Xiong L,Stevenson B,Zhu JK.Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis:interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways[J].Plant Cell,1997,9:1935-1949
35 GmblerΜ,Hoffman BJ.A simple and very elflent method for generating cDNA library[J].Gene,1983,25:263-269
36许兰珍,何永睿,姜国金,刘小丰,陈善春.cDNA文库构建及其在植物抗性研究中的应用[J].安徽农业科学,2007,35(3):660-662,664
37 Carninci P,Shibata Y,Hayatsu N,Sugahara Y,Shibata K,Itoh M,Konno H,Okazaki Y , Muramatsu M , Hayashizaki Y.Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
38 Kato S,Sekine S,Oh SW,Kim NS,Μmezawa Y,Abe N,Yokoyama-Kobayashi M,Aoki T.Construction of a human full-length cDNA bank[J].Gene,1994,150(2):243-250
39 Seki M,Carninci P,Nishiyama Y,Hayashizaki Y and Shinozaki K.High-efficiency cloning Arabidopsis full-length cDNA by biotinylated CAP trapper[J].The plant Journal,1998,15(5):707-720
40 Suzuki Y,Sugano S.Construction of a full-length enriched and a 5’-end enriched cDNA library using the oligo-capping method[J].Methods Mol Biol,2003,221 (1):73-91
41 Suzuki Y,Yoshitomo-Nakagawa K,Maruyama K,Suyama A,Sugano S.Construction and characterization of a full-length-enriched and a 5’-end-enriched cDNA library[J].Gene,1997,200(122):149-156
42 Zhu YY,Machleder EM,Chenchik A,Li R,Siebert PD.Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction[J].Biotechniques,2001,30(4):892-897
43 Efimov VA,Chakhmakhcheva OG,Archdeacon J,Fernandez J,Fedorkin ON,Dorokhov Yu L,Atabekov JG.Detection of the 5’-cap structure of messenger RNAs with the use of the cap-jumping approach[J].Nucl Acids Res,2001,29(22):4751-4759
44 Carninci P,Shibata Y,Hayatsu N,Sugahara Y,Shibata K,ItohM,Konno H,Okazaki Y , Muramat su M , Hayashizaki Y.Nor-malization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
45 Carninci P,Kvam C,Kitamura A,Ohsumi T,Okazaki Y,Itoh M,Kamiya M,Shibata K,Sasaki N,Izawa M,Muramatsu M,Hayashizaki Y,Schneider C.High-efficiency full-length cDNA cloning by biotinylated CAP trapper[J].Genomics,1996,37 (3):327-336
46 Carninci P,Westover A,Nishiyama Y,Ohsumi T,Itoh M,Nagaoka S,Sasaki N,Okazaki Y,Muramat su M,Schneider C,Hayashizaki Y.High efficiency selection of full-length cDNA by improved biotinylated captrapper[J].DNA Res,1997,4(1): 61-66
47 Edery I,Chull,Sonenberg N,Pelletier J.An efficient strategy to isolate full-length cDNAs.based on an mRNA cap retention procedure(CAPture)[J].Mol Cell Biol,1995,15(6):3363-3371
48 Sugahara Y , Carninci P , Itoh M , etal.Comparative evaluation of 5’-end-sequence quality of clones in CAP trapper and other full-length-cDNA libraries[J].Gene,2001,263:93-102
49 Maruyama K,Sugano S.Oligo-capping:a simple method to replace the cap structure of eukaryotic mRNAs with oligoriboucleotides[J].Gene,1994,138(1-2):171-174
50 Suzuki Y,Yoshitomo-Nakagawa K,Maruyama K,Suyama A, Sugano S. Construction and characterization of a full length-enriched and a 5’-end-enriched cDNA library[J].Gene,1997,200(1-2):149-156
51 Suzuki Y,Sugano S.Construction of full-length-enriched cDNA libraries.The oligo-capping method[J].Methods Mol Biol,2001,175:143-153
52 Suzuki Y,Sugano S.Construction of a full-length enriched and a 5’-end enriched cDNA library using the oligo-capping method[J].Methods Mol Biol,2003,221:73-91
53 Chenchik A,Moqadam F.Siebert P.RNA:Isolation,Analysis and Synthesis[M].New York:Wiley-Liss,l996,273-32l
54毛新国,景蕊莲,孔秀英,赵光耀,贾继增.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7):865-873
55 Zhu YY,Machleder EM,Chenchik A,Li R,Siebert PD.Reverse transcriptase template switching:a SMART approach for full-length cDNA library construction[J]. Biotechniques,2001,30(4):892-897
56 Wellenreuther R,Schupp I,Poustka A,etal.The German cDNA consortium SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones[J].BMC Genomics,2004,36(5):1-8
57董志敏,张宝石,关志霞,常汝镇,邱丽娟.全长cDNA文库的构建方法[J].中国农学通报,2007,22(2):51-53
58王峰,王金发.水稻幼叶及幼穗cDNA文库的构建及初步分析[J].中山大学学报,2005,44(2):74-77
59董海滨,管荣展.双低油菜华双3号幼苗全长cDNA文库的构建[J].南京农业大学学报2005,28(3):123-125
60徐碧玉,苏伟,张建斌,刘菊华,金志强.香蕉果实SMART eDNA文库的构建及利用PCR方法筛选香蕉Actin2基因[J].热带亚热带植物学报2005,13(5):375-380
61蔡勤安,王玉成,张道远,董玉芝.渗透胁迫下白花柽柳cDNA文库的构建及EST分析[J].生物技术,2005,15(3):22-26
62 Carninci P,Nishiyama Y,Westover A,Itoh M,Nagaoka S,Sasaki N, Okazaki Y,Muramatsu M , Hayashizaki Y.Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA[J].Proc Natl Acad Sci USA,1998,95(2):520-524
63 Carninci P,Hayashizaki Y.High-efficiency full-length cDNA cloning[J].Methods Enzymol,1999,303:19-44
64 Carninci P,Shiba a Y,Hayatsu N,Sugahara Y,Shibata K,Itoh M,Konno H,Okazaki Y , Muramatsu M , Hayashizaki Y.Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes[J].Genome Res,2000,10(10):1617-1630
65 Carninci P,Waki,Sh ki T,Konno H,Shibata K,Itoh M,Aizawa K,Arakawa T,Ishii Y , Sasaki D , Hayashizaki Y.Targeting a complex transcriptome:the construction of the mouse full-length cDNA encyclopedia[J].Genome Res, 2003,13(6B):1273-1289
66毛新国,孔秀英,赵光耀,贾继增.利用改进的Cap-trapper法构建拟斯卑尔脱山羊草(Ae.speltoides Tausch)全长cDNA文库[J].遗传学报,2005,32 (8):811-817
67 Seki M,Narusaka M,Yamaguchi-Shinozaki K,etal.Arabidopsis encyclopedia using full-length cDNAs and its application.Plant Physiology and Biochemistry[J].2001,39:211-220
68 Seki M, Narusaka M, Ishida J, etal.Monitoring the expression profiles of 7000 Arabidopsis genes under drought,cold,and high-salinity stresses using a full-length cDNA microarray[J]. Plant J, 2002a,31:279-292
69 Seki M,Narusaka M,Kamiya A,et al.Functional annotation of a full-length Arabidopsis cDNA collection[J].Science,2002b,296:141-145
70 Seki M,Ishida J,Narusaka M,etal.Monitoring the expression pattern of around 7000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray[J].Funct.Integr.Genomic,2002c,2:282-291
71 Seki M , Satou M , Sakurai T , et al.RIKEN Arabidopsis full-length cDNA database[J].Trends Plant Sci,2002d,7:562-563
72 Kikuchi S,Satoh K,Nagata T,etal.Collection,mapping,and annotation of over 28000 cDNA clones from japonica Rice[J].Science,2003,301:376-378
73毛新国.小麦族A、S、O二倍体种全长cDNA文库构建及序列初步分析[D].中国农业科学院博士论文,2004
74 Osato N,Itoh M,Konno H etal.A computer-based method of selecting clones for a full-length cDNA project: simultaneous collection of negligibly redundant and variant cDNAs[J].Genome Res,2002,12:1127-1134
75 Kasuga M,Liu Q,Miura S,etal.Improving plant drought,salt,and freezing tolerance by gene transfer of a single Reymond P,Weber H,Damond M, Farmer EE,ifferential gene expression in response to mechanical wounding and insect feeding in Arabidopsis[J].Plant Cell,2000,12:717-719
76 Shinozaki K,and Yamaguchi-Shinozaki K.Gene expressionand signal transduction in water-stress response[J].Plant Physiology,1997,115:327-334
77 Wang R,Guegler K,LaBrie ST,etal.Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate[J].Plant Cell,2000,12:1491-1509
78 Ahn S,Anderson JA,Sorrells ME,etal.Homoeologous relationships of rice,wheatand maize chromosomes[J].Mol.Gen.Genet,1993a,241:483-490
79 Ahn S , and Tanksley SD.Comparative linkage maps of the rice and maize genomes[J].Proc.Natl.Acad.Sci.USA,1993b,90:7980-7984
80 Bennetzen JL,and Ma JX.The genetic colinearity of rice and other cereals on the basis of genomic sequence analysis[J].Curr.Opin.Plant Biol,2003,6:128-133
81 Swigonova Z , Lai J , Ma J , etal.Close split of sorghum and maize genome progenitors[J].Genome Res,2004,14:1916-1923
82李慧卿,马文元,李慧勇.沙冬青抗逆性研究进展[J].世界林业研究,2000,13(5):67-71
83陶玲,李新荣,刘新民,等.中国珍惜濒危荒漠植物保护等级的定量研究[J].林业科学,2001,37(1):52-57
84 Burke MJ,Gusta LV,Quam me HA.Freezing and injury in plants[J].Ann Rev Plant Physiol,1976,27:507-528
85梁秋霞,曹刚强,押辉远.抗冻蛋白在园艺植物遗传改良中的研究进展[J].北方园艺,2006 (6):50-52
86吴建民,幸华,赵志光,等.植物抗冻蛋白的研究进展及其应用[J].冰川冻土,2004,26(4):482-487
87周晓蕾,陈滔滔,王保怀,等.沙冬青抗冻蛋白热滞活性的DSC研究[J].物理化学学报,2001,17(1):66-69
88 Griffith M,Ala P,Yang DSC,etal.Antifreeze protein produced endogenously in winter rye leaves[J].Plant Physiol,1992,100:593–596
89费云标,孙龙华,黄涛,等.沙冬青高活性抗冻蛋白的发现[J].植物学报,1994,36 (8):649-650
90 Huang T,Duman JG.Purification and characterization of thermal hysteresis protein from cold-acclimated kale,Brassica oleracea [J].Cryobiology,1995,32:577-581
91 At1c1 ?,Nalbantolu B.Effect of apoplastic proteins on freezing to lerance in leaves[J].Phytochemistry,1999,50:755-761
92江勇,魏令波,费云标,等.分离和鉴定沙冬青抗冻蛋白质[J].植物学报,1999,41 (9):967-971
93魏令波,江勇,舒念红,等.沙冬青叶片热稳定抗冻蛋白特性分析[J].植物学报,1999,41(8):837-841
94费云标,魏令波,高素琴,等.沙冬青抗冻蛋白的分离、纯化及其理化特性分析[J].科学通报,2000,45(20):2185-2189
95舒念红,魏令波,高素琴,等.植物抗冻蛋白纯化、活性与二级结构测定[J].中国医学研究与临床,2003,1(12):45-47
96 Wang W,Wei L.Purification of boiling-soluble antifreeze protein from the legumeAmmopiptanthus mongolicus[J].Prep Biochem Biotechnol,2003, 33(1):67-80
97 Wang W,Wei L,Wang G.Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods[J].Chromatogr Sci,2003,41(9):489-493
98王维香.沙冬青抗冻蛋白的分离纯化及特性鉴定[D].大连理工大学博士论文, 2004
99王维香,魏令波,张虎.沙冬青热稳定抗冻蛋白的分离纯化及其部分性质研究[J].四川大学学报(自然科学版),2007,44(4):912-917
100 Fei YB, Cao PX, Gao SQ, etal.Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus[J].Prep Biochem Biotechnol,2008,38(2):172-183
101鲁春芳,尹林克,牟书勇,等.新疆沙冬青叶片氨基酸和蛋白质的测试与分离以及抗冻蛋白的鉴定结果分析[J].武汉植物学研究,2007,25(5):531-534
102 Yun-Biao Fei, Peng-Xiu Cao, Su-Qin Gao,etal.Discovery of a Homolog of Siderophilin in a Plan[J]. Journal of integrative plant biology , 2005 ,47(12):1409-1411
103 Liu M,Shen X,Yin W,etal.Functional analysis of cold-inducible cDNA clones in the legume Ammopiptanthus mongolicus[J].Cryo Letters, 2005,26(4):213-222
104 Liu MQ , Lu CF , Shen X et al.Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus[J].DNA Sequence,2006,17(5):342-349
105刘美芹,沈昕,卢存福,等.一种改进的固相扣除杂交法直接克隆全长差异表达基因[J].北京林业大学学报,2007,29(5):67-72
106陈奕吟.沙冬青低温诱导基因转化烟草及转基因植物的抗寒性研究[D].北京林业大学硕士学位论文,2007
107孙燕.沙冬青CBL1基因的克隆及表达特性研究[D].北京林业大学硕士学位论文,2005
108 Batistic O, Kudla J.Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J].Planta 2004,219: 915-924
109孙芳.沙冬青CBLI基因的功能研究[D].北京林业大学硕士学位论文,2009
110宋健.沙冬青耐寒基因AmGolS的克隆和遗传转化研究[D].山东农业大学硕士学位论文,2007
111郭九峰,孙国琴,沈传进,等.沙冬青cDNA文库的构建和EST分析[J].华北农学报,2007,22(4):37-41
112乔慧蕾,王瑞刚,郭九峰,等.沙冬青脱水素基因的克隆及表达载体的构建[J].华北农学报,2009,(4):89-90
113 Pengxiu Cao,Jian Song,Chunjiang Zhou,etal.Characterization of multiple coldinduced genes from Ammopiptanthus mongolicus and functional analyses of gene AmEBP1[J].Plant Molecule Biology,2008,529-539
114张晗.棉花CBF调控因子在逆境条件下的表达调控及沙冬青CBF基因的克隆.中国农业科学院硕士学位论文,2008
115刘静,王长宪,王斌,等.抗寒基因AmGS高效转化体系的研究[J].山东农业大学学报(自然科学版),2009,40(2):191-194
116 Sambrook,分子克隆实验指南[M].北京:科学出版社,2002,第三版
117李宏,王新力.植物组织RNA提取的难点及对策[J].生物技术通报,1999,1:36-39
118王玉成,杨传平,姜静.木本植物组织总RNA提取的要点与原理[J].东北林业大学学报,2002,30:1-4
119朱昀,王猛,贾志伟,练云,金颖,王国英.一种从富含多糖的玉米幼穗中提取RNA的方法[J].植物学通报,2007,24(5):624-628
120周春娥,段红英,齐力旺.木本植物老根老叶总RNA的提取方法[J].安徽农业科学,2007,35:1831-2832
121高鹏.玉米幼苗淹水诱导表达基因的克隆及其功能分析[M].北京,中国农业大学博士论文,2003
122 Selenge D,Batsuren D,Batriov E Kh,etal. Chemical study of the flora of mongolicus isoflavones of Ammopiptanthus mongolicus[J].Khim Prirodn Soedin.1986,2:242
123田晓明,陈世忠,屠鹏飞,雷连娣.沙冬青地上部分的化学成分研究[J].中国中药杂志.2008,33:10
124 Ainsworth C.Isolation of RNA from floral tissue of Rumex acetosa (sorrel)[J].Plant Mol Biol Rep,1994,12:198-203,212-215
125王昆,王颖,鲍永利,孟祥颖,乌垠,易静文,李玉新.人参叶全长cDNA文库的构建及部分克隆的序列分析[J].分子科学学报,2006,22(1):58-61
126董志敏,李英慧,张宝石,关荣霞,常汝镇,邱丽娟.一种获得大片段克隆的SMART全长cDNA文库构建方法[J].大豆科学,2007,3:223-227
127 Zheng J,Zhao J,Tao Y,Wang J,Liu Y,Fu J,Jin Y,Gao P,Zhang J, Bai Y,Wang G.Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray[J].Plant Mol. Biol,2004,55(6):807-823
128张锦鹏.谷子在干旱逆境中差异表达基因的分离与表达谱分析[D].中国农业大学博士学位论文,2006