斑茅抗逆性评价及其BADH基因的克隆表达
|
摘要
斑茅(Erianthus arundinaceus)是甘蔗的重要近缘属植物，生长势强、抗旱、耐瘠、宿根性好，是甘蔗育种的重要基因资源。本研究在系统分析斑茅抗逆性基础上，通过同源克隆结合RACE技术、Northern杂交、原核表达等，克隆斑茅BADH基因，并研究其在抗逆方面的分子作用。结果如下：
在干旱和盐胁迫条件下，斑茅丙二醛含量增幅较拔地拉小，质膜受损小，抗氧化酶类的清除、防御膜脂过氧化的能力强、脯氨酸积累多，斑茅的抗逆性明显强于拔地拉；利用常规比色法测定拔地拉和斑茅中的甜菜碱含量，表明斑茅甜菜碱含量高达10.73 mg／100g FW，与已测定的15植物甜菜碱含量相比，斑茅的甜菜碱含量仅次于藜科植物碱蓬和盐吸，位居第三，而拔地拉不含有或痕迹量甜菜碱。
用RT-PCR和RACE技术克隆了斑茅甜菜碱醛脱氢酶基因。斑茅BADH cDNA全长1718bp，5′端290bp左右片段GC含量高达75.9％，包含一个1515 bp的开放读码框，5′端非编码区35bp，3′端非编码区165bp，理论分子量为54.905 kDa，其推演氨基酸含有十肽VTLELGGKSP和一个与酶功能有关的Cys为醛脱氢酶高度保守的序列，以及C-端信号肽SKL。将斑茅BADH编码区1515 bp核苷酸序列在GenBank中进行比较，发现其与Zea mays PCO110216的已知部分序列同源性为97％；与S.bicolor BADH1部分序列同源性为95％；与O.sativa BADH、H.vulgare BBD2和T.aestivum BADH的5′端无同源性。
分子进化分析表明，22个BADH基因由同一基因进化成五个类群，双子叶植物进化变异较大；所有已知禾本科植物斑茅、高粱、玉米、大麦、小麦的BADH基因为一簇，其中按同工酶类型分为两组；斑茅与高粱的同源性高于斑茅与玉米，以及高粱与玉米的同源性。
包含醛保守序列的斑茅BADH部分cDNA序列作探针，进行Southern和Northern杂交分析。结果表明，斑茅BADH在基因组中有一到两个拷贝；斑茅BADH为诱导型表达基因，干旱胁迫，表达量增加，复水后其表达量减少；盐胁迫时，表达量随着胁迫时间增加，然后保持某一水平，但又受环境温度影响；盐胁迫10天时，根中甜菜碱醛脱氢酶的表达量大于茎中的，茎中的大于叶中。
构建斑茅甜菜碱醛脱氢酶的原核表达载体，原核表达斑茅甜菜碱醛脱氢
福建农林大学博士学位论文
酶基因，在酶粗提液中检测到酶的比活力为9.04Inn。功吐n一，m扩蛋白。
斑茅BADH基因的成功分离并转导甘蔗将有利于实现甘蔗抗早基因工程，
提高抗早育种效率。
Erianthus arundinaceus, the related genara of Saccharum, has good applicability, stress resistance, vigour and ratooning ability. So, it is important gene resource for sugarcane breeding. Based on the comparative analysis for stress resistance of E. arundinaceus and Saccharum, BADH gene from E. arundinaceus was cloned and its expression was analysed in E.coli or under high salinity and drought stress. The results were as follows:
The increments of MDA content and the plasma membrane permeability in leaves of E.arundinaceus were less than those in Badila (S. qfficinarum L.) subjected to water deficit or salt stress, and higher activities of SOD and POD in leaves of E.arundinaceus. 10.73 mg/100g FW of glycinbetaine in water-stressed E.arundinaceus was less than that of S. glauca or S. ussuriensis, but higher than those of the other reported plants. No glycine betaine accumulated in water-deficit Badila.
The full-length BADHcDNA from E.arundinaceus was isolated by RT-PCR and rapid amplified cDNA ends (RACE). E.arundinaceus BADH was 1718 bp in length, including 35 bp 5'UTR, 165 bp 3'UTR and 1515-base-pair open reading frame (ORF) encoding protein of 54.905 kDa. 75.9% of G and C was found in the 290bp length of its 5'end. The deduced amino acid sequence of E.arundinaceus BADH included a decapeptide sequence "VTLELGGKSP" and a Cysteine residue, which is highly conserved among general aldehyde dehydrogenases (ALDH). It also contained a tripeptide SKL at the C-terminnal, a signal known to target to microbodies. The BLAST analysis in GenBank showed that overall coding region of E. arundinaceus BADH is c. 97% homologous to the Zea mays cDNA sequence ( PCO110216), c. 95% to the partial cDNA of Sorghum bicolor BADH1. However, its 5'end had no homology to O. sativa BADH, H. vulgare BBD2 and T. aestivum BADH.
The phylogenetic analysis from 22 kinds of plant BADHs indicated the larger variability in the dicotyledonous plants, one group in the Gramineae plants including BADH genes form E.arundinaceus, S.vulgare, Zea mays, H. vulgare and
T. aestivum, which branches out into two groups corresponding to betaine aldehyde dehydrogenase isozymes..
Southern and Northern blot analysis were carried out using cDNA fragment of the conserved domain sequence of E.arundinaceus BADH as a probe. Southern blot showed one or two copies of E.arundinaceus BADH in the genome of E. arundinaceus. Northern blot showed the E. arundinaceus BADH was expressed at high levels under drought stress, then decreased after recovering water. However, its expression levels increased with the elongation of salt stress duration, then kept at a certain level. On the tenth day of salt stress, the level of mRNA for Karundinaceus BADH in stem of E. arundinaceus was higher than that in leaves, lower in roots.
The expression vector of E.arundinaceus BADH was constructed. E.arundinaceus BADH could be expressed in E.coli BL21.The specific activity of E.arundinaceus BADH induced by IPTG was 9.04 nmolmin-1mg-1protein.
Cloning of E. arundinaceus BADH gene and its successful transformation into sugarcane cultivars will be an alternative to improve drought tolerance in sugarcane in future.
在干旱和盐胁迫条件下，斑茅丙二醛含量增幅较拔地拉小，质膜受损小，抗氧化酶类的清除、防御膜脂过氧化的能力强、脯氨酸积累多，斑茅的抗逆性明显强于拔地拉；利用常规比色法测定拔地拉和斑茅中的甜菜碱含量，表明斑茅甜菜碱含量高达10.73 mg／100g FW，与已测定的15植物甜菜碱含量相比，斑茅的甜菜碱含量仅次于藜科植物碱蓬和盐吸，位居第三，而拔地拉不含有或痕迹量甜菜碱。
用RT-PCR和RACE技术克隆了斑茅甜菜碱醛脱氢酶基因。斑茅BADH cDNA全长1718bp，5′端290bp左右片段GC含量高达75.9％，包含一个1515 bp的开放读码框，5′端非编码区35bp，3′端非编码区165bp，理论分子量为54.905 kDa，其推演氨基酸含有十肽VTLELGGKSP和一个与酶功能有关的Cys为醛脱氢酶高度保守的序列，以及C-端信号肽SKL。将斑茅BADH编码区1515 bp核苷酸序列在GenBank中进行比较，发现其与Zea mays PCO110216的已知部分序列同源性为97％；与S.bicolor BADH1部分序列同源性为95％；与O.sativa BADH、H.vulgare BBD2和T.aestivum BADH的5′端无同源性。
分子进化分析表明，22个BADH基因由同一基因进化成五个类群，双子叶植物进化变异较大；所有已知禾本科植物斑茅、高粱、玉米、大麦、小麦的BADH基因为一簇，其中按同工酶类型分为两组；斑茅与高粱的同源性高于斑茅与玉米，以及高粱与玉米的同源性。
包含醛保守序列的斑茅BADH部分cDNA序列作探针，进行Southern和Northern杂交分析。结果表明，斑茅BADH在基因组中有一到两个拷贝；斑茅BADH为诱导型表达基因，干旱胁迫，表达量增加，复水后其表达量减少；盐胁迫时，表达量随着胁迫时间增加，然后保持某一水平，但又受环境温度影响；盐胁迫10天时，根中甜菜碱醛脱氢酶的表达量大于茎中的，茎中的大于叶中。
构建斑茅甜菜碱醛脱氢酶的原核表达载体，原核表达斑茅甜菜碱醛脱氢
福建农林大学博士学位论文
酶基因，在酶粗提液中检测到酶的比活力为9.04Inn。功吐n一，m扩蛋白。
斑茅BADH基因的成功分离并转导甘蔗将有利于实现甘蔗抗早基因工程，
提高抗早育种效率。
Erianthus arundinaceus, the related genara of Saccharum, has good applicability, stress resistance, vigour and ratooning ability. So, it is important gene resource for sugarcane breeding. Based on the comparative analysis for stress resistance of E. arundinaceus and Saccharum, BADH gene from E. arundinaceus was cloned and its expression was analysed in E.coli or under high salinity and drought stress. The results were as follows:
The increments of MDA content and the plasma membrane permeability in leaves of E.arundinaceus were less than those in Badila (S. qfficinarum L.) subjected to water deficit or salt stress, and higher activities of SOD and POD in leaves of E.arundinaceus. 10.73 mg/100g FW of glycinbetaine in water-stressed E.arundinaceus was less than that of S. glauca or S. ussuriensis, but higher than those of the other reported plants. No glycine betaine accumulated in water-deficit Badila.
The full-length BADHcDNA from E.arundinaceus was isolated by RT-PCR and rapid amplified cDNA ends (RACE). E.arundinaceus BADH was 1718 bp in length, including 35 bp 5'UTR, 165 bp 3'UTR and 1515-base-pair open reading frame (ORF) encoding protein of 54.905 kDa. 75.9% of G and C was found in the 290bp length of its 5'end. The deduced amino acid sequence of E.arundinaceus BADH included a decapeptide sequence "VTLELGGKSP" and a Cysteine residue, which is highly conserved among general aldehyde dehydrogenases (ALDH). It also contained a tripeptide SKL at the C-terminnal, a signal known to target to microbodies. The BLAST analysis in GenBank showed that overall coding region of E. arundinaceus BADH is c. 97% homologous to the Zea mays cDNA sequence ( PCO110216), c. 95% to the partial cDNA of Sorghum bicolor BADH1. However, its 5'end had no homology to O. sativa BADH, H. vulgare BBD2 and T. aestivum BADH.
The phylogenetic analysis from 22 kinds of plant BADHs indicated the larger variability in the dicotyledonous plants, one group in the Gramineae plants including BADH genes form E.arundinaceus, S.vulgare, Zea mays, H. vulgare and
T. aestivum, which branches out into two groups corresponding to betaine aldehyde dehydrogenase isozymes..
Southern and Northern blot analysis were carried out using cDNA fragment of the conserved domain sequence of E.arundinaceus BADH as a probe. Southern blot showed one or two copies of E.arundinaceus BADH in the genome of E. arundinaceus. Northern blot showed the E. arundinaceus BADH was expressed at high levels under drought stress, then decreased after recovering water. However, its expression levels increased with the elongation of salt stress duration, then kept at a certain level. On the tenth day of salt stress, the level of mRNA for Karundinaceus BADH in stem of E. arundinaceus was higher than that in leaves, lower in roots.
The expression vector of E.arundinaceus BADH was constructed. E.arundinaceus BADH could be expressed in E.coli BL21.The specific activity of E.arundinaceus BADH induced by IPTG was 9.04 nmolmin-1mg-1protein.
Cloning of E. arundinaceus BADH gene and its successful transformation into sugarcane cultivars will be an alternative to improve drought tolerance in sugarcane in future.
引文
1．陈如凯，林彦铨，张木清，等．现代甘蔗育种的理论与实践．北京：中国农业出版社，2003
2．扬洪强，贾文锁，张大鹏．植物水分胁迫信号识别与转导．植物生理学通讯，2001，37(2)：149～154
3．缪颖，伍炳华．植物抗逆性的获得与信息传导．植物生理学通讯，37(1)：71～76
4．颜华，贾良辉，王根轩．植物水分胁迫诱导蛋白的研究进展．生命的化学，2002，22(2)：165～168
5．余光辉，李玲，曾福华．水分胁迫的基因表达和信号转导．亚热带植物科学，2002，31(1)：57～62
6. Shinozaki K, Yamaguchi-Shinozaki K, Mizoguchi T, et al. Molecular responses to water stress in Arabilopsis thaliana. J Plant Res, 1998, 111:345～351
7. Bray E A. Plant responses to water deficit. Trends Plant Sci, 1997, 2: 48～54
8. Meskiene I, Heft H. MAP kinase pathways: molecular plug-and-play chip for the cell. Plant Mol Biol, 2000, 42: 791～806
9. Mikami K, Katagiri T, Iuchi S, et al. A gene encoding phosphatidylinositol-4-phosphate 5-kinase is induced by water stress and abscisic acid in Arabidopsis thsliana.Plant J, 1998, 15(4): 563～568
10. Takahashi S, Katagiri T, Yamaguchi-Shinozaki, et al. An Arabidopsis gene encoding a Ca~(2+)-binding protein is induced by Abscisic Acid during dehydration. Plant and Cell Physiology, 2000, 41(7): 898～903
11. Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca~(2+)-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J, 2000, 23: 319～327
12. Uno Y, Furihata T, Abe H, et al. Arabidopsis basic leucine zipper transcription factors involved an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc Natl Acad Sci USA, 2000,97: 11632～11637
13. Liu Q, Kasuga, M, Sakuma Y, et al. Two transduction 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 Arabilopsis. Plant Cell, 1998, 10: 1391～1406
14. Nakashima K, Kiyosue T, Yamaguchi-Shinozaki K, et al. A nuclear gene erdl encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally upregulated during senescence in Arabidopsis thaliana. Plant J, 1997, 12(4): 851～861
15．刘强，张贵友，陈受宜．植物转录因子的结构与调控作用．科学通报，2000，45(14)：1465～1474
16．经艳芬，桃联安，扬李和，等．云南野生甘蔗种质血缘F_3代亲本材料抗旱性研究初报．甘蔗，2002，9(1)：19～23
17．陈能武，扬荣仲，黄久凯，等．甘蔗抗旱性鉴定方法的研究：1．甘蔗苗期抗旱性鉴定方法的筛选．四川甘蔗，1992，3(4)：5～9
18．张木清，陈如凯，余松烈．水分胁迫下蔗叶活性氧代谢的数学分析．作物学报，1996，22(6)：729～735
19．文建成，陈学宽，符菊芬，等．质膜透性与丙二醛(MDA)含量的变化评价甘蔗品种抗旱性初探．甘蔗，1998，5(3)：1～5
20．陈如凯，张木清，陆裔波．干旱胁迫对甘蔗生理影响的研究．甘蔗，1995，2(1)：1～6
21．高三基，罗俊，陈如凯，等．甘蔗品种抗旱性光合生理指标及其综合评价．作物学报，2002，28(1)：94～98
22．吴才文，陈能武，扬荣仲，等．水分胁迫及复水对甘蔗生长发育及相关生理指标的影响．甘蔗，1998，5(3)：6～12
23．徐永胜．甘蔗愈伤组织对水分胁迫的生理反应及其抗旱性．广西农学院硕士论文，1986
24. Rap C K, Asckan S. Studies on free proline association to drought resistance in sugarcane. The Sugar J, 1978, (1): 23～24
25. Rutherford R S. The assessment of profile accumulation as a mechanism of drought resistance in sugarcane. Prec S Afric Sug Tech Asseci, 1989: 136 141
26．潘世明．甘蔗抗旱性直接鉴定法的综合评价．福建甘蔗，1995，(1)：25～28
27. Inman-Bamber N G, Thompson G D. The reaction of two varieties of sugarcane to water stress. Field Crop Research, 1986, (14):15～28
28．陈少裕．甘蔗抗旱机理研究现状与展望．福建农学院学报，1991，20(1)：12～17
29．张木清，陈如凯，余松烈．水分胁迫下蔗叶多胺代谢变化及其同抗旱性的关系．植物生理学报，1996，22(3)：327～332
30. Kasuga M, Liu Q, YamaguchiS K, et al. Improving plant drought, salt tolerance by gene transfer of a single stress-inducible transporter factor. Nature Biotechnol, 1999, 17: 287～291
31. Bowler C, Slooten L, Vandenbranden S, et al. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J, 1991, 10: 1723～1732
32. Gupta A S, Heinen J L, Holaday A S, et al. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA, 1993, 90: 1629～1633
33. Dure L Ⅲ, Greenway S C, Galau G A. Developmental biochemistry of cotton seed embryogenesis and germination. ⅪⅤ. Changing mRNA populations as shown by in vitro and in vivo protein synthesis. Biochemistry, 1981, 20: 4162～4168
34. Kishor P B K, Hong Z, Miao G H, et al. Overexpression of [delta] -Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants. Plant Physiol, 1995, 108: 1387～1394
35. Holmstrom K O, Mantyla E, Welin B, et al. Drought tolerance in tobacco. Nature, 1996, 379: 683～684
36. Pilon-Smits E A H, Ebskamp M J M, Paul M J, et al. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol, 1995, 107: 125～130
37. Sheveleva E, Chmara W, Bohnert H J, et al. Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L. Plant Physiol, 1997, 115: 1211～1219
38. Capell T, Escobar C, Lui H, et al. Over-expression of the oat arginine decarboxylase cDNA in transgenic rice(Oryza sativa L.)affects normal development patterns in vltro and results in putrescine accumulation in transgenic plants. Theor Appl Genet, 1998, 97: 246～254
39. Zhu B, Su J, Chang M, et al. Overexpression of a D-pyrroline-5-carboxylate
synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice. Plant Sci, 1998, 139: 41～48
40．王自章．农杆菌介导海藻糖合酶基因遗传转化甘蔗研究．福建农林大学博士学位论文，2001
41. Falco M C. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell Report, 2000, 19(12): 301～304
42. Arencibia A, Elva, Chan, MT, et al. An efficient protocol for sugarcane (Saccharum officinarum L.)Transformation mediated by Agrobacterium tumefaciens. Transgenic Research, 1998, 7:213～222
43．田允波，葛长荣．甜菜碱在畜牧生产中的应用．内蒙古畜牧科学，1998，19(2)：10～13
44．刘建中，扬晓建，将宗勇．甜菜碱在饲料中的应用．广东饲料，1999，(5)：12～23．
45．钟伟泽．甜菜碱在饲料工业中的应用．广东饲料，1998，(5)：28～29．
46．宋丽丽，阚振荣．甜菜碱的制备、含量测定及其应用．河北林果研究，2002，17(4)：382～388
47. Weissensteiner T, Lanchbury J S. Stratergy for controlling preferential amplification and avoiding false negatives in PCR typing. Biotechniques, 1996, 21: 1102～1108
48. Henke W, Herdel K, Jung K, et al. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res, 1997,25, 3957～3958
49．梁峥，骆爱玲．甜菜碱和甜菜碱合成酶．植物生理学通讯，1995，31(1)：1～8．
50. Wyn Jones R G, Storey R. Physiology and biochemistry of drought resistance in plants. New York: Academic Press, 1981.
51. McNeil S D, Nuccio M L, Hanson A D. Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistance. Plant Physiol, 1999, 120: 945～949
52. Grumet R, Hanson A D. Genetics evidence of an osmoregulary function of glycinebetaine accumulation in barley. J Plant Physiol, 1986, 13: 353～364
53. Guy R D, Warne P G, Reid D M, et al. Glycinebetaine content of halophytes: improved analysis by liquid chromatography and interpretations of results. Physiol Plant, 1984, 61: 195～202
54．梁峥，赵原，汤岚，等．甜菜碱对呼吸酶的保护作用．植物学报．1994，36(12)：947～951．
55．侯彩霞，於新建，李荣，等．甜菜碱稳定PSⅡ放氧中心外周多肽机理．中国科学(C辑)，1998，28(4)：355～361．
56．张士功，高吉寅，宋景芝．甜菜碱对NaCl胁迫下小麦细胞保护酶活性的影响．植物学通报，1999，16(4)：429～432
57．孙文越，王辉，黄久长．外源甜菜碱对干旱胁迫下小麦幼苗膜脂过氧化作用的影响．西北植物学报，2001，21(3)：487～491
58．赵博生，衣艳君，刘家尧．外源甜菜碱对干旱／盐胁迫下的小麦幼苗生长和光合功能的改善．植物学通报，2001，18(3)：378～380
59．衣艳君，刘家尧，骆爱玲，等．转BADH基因烟草的光系统Ⅱ和呼吸酶活性的变化．植物学报，1999，41(9)：993～996
60. Nomura M, Hibino T, Takabe T, et al. Transgenically produced glycinebetaine protects ribulose-1, 5-bisphosphate carboxylase/oxygenase from inactivation in Synechococcus sp. PCC7942 under salt stress. Plant Cell Physiol, 1998, 39(4): 425～432
61．侯彩霞，汤章程．细胞相容性物质的生理功能及作用机制．植物生理学通讯，1999，35(1)：1～7．
62．许雯，孙梅好，朱亚芳，等．甘氨酸甜菜碱增强青菜抗盐作用．植物学报，2001，43(8)：809～814
63. Brouquisse R, Weigel P, Rhodes D, et al. Evidence for ferredoxin-depedent choline monooxygenase from spinach chloroplast stroma. Plant Physiol,1989, 90: 322～329.
64. Burnet M, Lafontaine P J, Hanson A D. Assay, purification, and partial characterzation of choline monooxygenase from spinach. Plant Physiol, 1995, 108: 581～588.
65. Rathinasabapathi B, Burnet M, Russell B L, et al. Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. Proc Natl Acad Sci USA, 1997, 94(7): 3454～3458.
66. Russell B L, Rathinasabapathi B, Hanson A D. Osmotic stress induces
expression of choline monooxygenase in sugar beet and amaranth. Plant Physiol, 1998, 116(2): 859～865.
67．沈义国，杜保兴，张劲松，等．山菠菜胆碱单氧化物酶基因(CMO)的克隆与分析．生物工程学报，2001．17(1)：1～6．
68. Meng Y L, Wang Y M, Zhang B, et al. Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions. Cell Res, 2001, 11(3): 187～193.
69. Nuccio M L, Russell B L, Nolte, K D, et al. The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J, 1998,16: 487～496.
70. Nuccio M L, McNeil S D, Ziemak M J, et al. Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway. Metab Eng, 2000, 2: 300～311.
71. McNeil S D, Rhodes D, Russell B, et al. Metabolic modeling identifies key constraints on an engineered glycinebetaine synthesis pathway in tobacco. Plant Physiol, 2000, 124: 153～162.
72．李秋莉 刘大伟，高晓蓉，等．辽宁碱蓬胆碱单加氧酶基因克隆及转基因烟草的耐盐性．Acta Botanica Sinica，2003，45(2)：242～247
73. Pan S M, Moreau R A, Yu C, et al. Betaine accumulation and betaine aldehyde dehydrogenase in spinach leaves. Plant Physiol, 1981, 67: 1105～1108.
74. Weigel P, Weretilnyk E A. Betaine aldehyde oxidation by spinach chloroplasts. Plant Physiol, 1986, 82: 753～759.
75. Arakawa K, Takabe T, Sugiyama T, et al. Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. J Biochem(Tokyo), 1987,101(6):1485～1488.
76. Weretilnyk E A, Hanson A D. Betaine aldehyde dehydrogenase polymorphism in spinach: genetic and biochemical characterization. Biochem Genet, 1988, 26(1-2): 143～151.
77. Weretilnyk E A, Hanson A D. Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity. Arch Biochem Biophys, 1989, 271(1): 56～63.
78. Valenzuela-Soto E M, Munoz-Clares R A. Purification and properties of betaine aldehyde dehydrogenase extracted from detached leaves of Amarathus hypochondriacus L. subjected to water deficit. J. Plant Physiol, 1994, 143:145-152
79. Arakawa K, Mizuno K, Kishitani S, et al. Immunological studies of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol, 1992, 33(7): 833～840.
80. Liang Z, Luo A L, Tang L, et al. Betaine aldehyde dehydrogenase from spinach leaves: purification and preparation of its antibody. Chinese J Bot, 1993, 5(1): 58～64.
81. Liang Z, Li Y, Shao Y,et al. Studies on some properties of betaine aldehyde dehydrogenase from spinach. Chinese J Bot, 1993, 5(2): 154～158.
82. Ana M G, Elisa M V. Porcine kidney betaine aldehyde dehydrogenase: purification and properties. Comparative Biochemistry and Physiology(Part B 119), 1998,119: 485～491.
83. Roberto V G, Carlos M J, Guillermo M H, et al. Rapid purification and properties of betaine aldehyde dehydrogenase from pseudomonas aeruginosa. Journal of Bacteriology, 1999, 181(4): 1292～1300.
84. Hibino T, Meng Y-L, Kawamitsu Y, et al. Molecular cloning and functional characterization of two kinds of betaine aldehyde dehydrogenase in betaine accumulating mangrove Avicennia marina(Forsk.) Vierh. Plant Mol Bio, 2001,45(3):353～363.
85. Falkenberg P, Strom A R. Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli. Biochim Biophys Acta, 1990, 1034:253～259.
86. Weretilnyk E A, Hanson A D. Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc Natl Acad Sci USA, 1990, 87(7): 2745～2749.
87. McCue K F, Hanson A D. Salt-inducible betaine aldehyde dehydrogenase form sugar beet: cDNA cloning and expression. Plant Mol Biol, 1992, 18: 1～11.
88．肖岗，张耕耘，刘凤华等．山菠菜甜菜碱醛脱氢酶基因研究．科学通报．1995，40(8)：741～745．
89. Legaria J, Rajsbaum R, Munoz-Clares R A, et al. Molecular characterization
of two genes encoding betaine aldehyde dehydrogenase from amaranth. Expression in leaves under short-term exposure to osmotic stress or abscisic acid. Gene, 1998,218(1-2):69～76.
90. Nakamura T, Nomura M, Mori H, et al. An isozyme of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol, 2001,42(10), 1088～1092
91. Wood A J, Saneoka H, Rhodes D, et al. Betaine aldehyde dehydrogenase in sorghum. Plant Physiol. 1996, 110(4): 1301～1308.
92. Nakamura T, Yokota S, Muramoto Y,et al. Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes. Plant J, 1997, 11(5): 1115～1120
93. Yong-Hua L, Qi Z. The sequence of Triticum aestivum Betaine Aldehyde Dehydrogenase Gene WBADH. Journal of Plant Physiology and Molecular Biology, 2002, 28(6): 495～496
94．舒卫国，艾万东，陈受宜．菠菜甜菜碱醛脱氢酶基因全序列分析．科学通报，1997，42(22)：2241～2245．
95. Brauner F, ebela M, Snégaroff J, et al. Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues. Plant Physiology and Biochemistry, 2003, 41: 1-10
96. Rathinasabapathi B, McCue K F, Gage D A, et al. Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta, 1994, 193(2): 155～162.
97．梁峥，马德钦，汤岚，等．菠菜甜菜碱醛脱氢酶基因在烟草中的表达．生物工程学报，1997，13(3)：236～240．
98．刘凤华，郭岩，谷冬梅，等．转甜菜碱醛脱氢酶基因植物的耐盐性研究．遗传学报，1997，24(1)：54～58．
99．郭岩，张莉，肖刚，等．甜菜碱醛脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究．中国科学，1997，27(2)：151～155．
100．郭北海，张艳敏，李洪杰，等．甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达．植物学报，2000，42(3)：279～283．
101．李银心，常凤启，杜立群，等．转甜菜碱醛脱氢酶基因豆瓣菜的耐盐性．植物学报，
2000，42(5)：480～484．
102．衣艳军，刘家尧，骆爱玲，等．转BABE基因烟草的光系统Ⅱ和呼吸酶活性变化．植物学报，1999，41(9)：993～996．
103. Kishitani S, Takanami T, Suzuki, M, et al. Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley. Plant Cell Environ, 2000,23:107～114
104．梁广坚．甜菜碱可以提高植物对低温的忍受力．肇庆学院学报，2003，24(2)．36～40，55
105. Diatchenko L, Lau Y F C, Campbell A P, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93:6025～6030
106. Siebert P D, Chenchik A, Kellogg DE, et al. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res, 1995, 23: 1087～1088
107. Wu G S, Saftig P, Peters C, et al. Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity. Oncogene, 1998, 16(17): 2177～2183
108. Peng L, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerse chain reaction. Science, 1992, 257: 967～970
109. Lisitsyn N, LisitsynN, Wigler M. Cloning the differences between two complex genomes. Science, 1993, 259: 946～951
110. Hubank M, Schatz D G. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res, 1994,22:5640～5648
111. Matz M, Usman N, Shag D, et al. Ordered differential display: a simple method for systematic comparison of gene expression profiles. Nucleic Acids Res, 1997, 25(12): 2541～2542
112. Broude N E, Chandra A, Smith CL. Differential display of genome subsets containing specific interspersed repeats. Proc Nail Acad Sci USA, 1997, 94(9): 4548～4553
113. Ivanova N B, Belyavsky A V. Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting. Nucleic Acids Res, 1995,23:2954～2958
114. Prashar Y, Weissman S M. Analysis of gene expression by display of 3'end restriction fragments of cDNA. Proc Natl Acad Sci USA, 1996,93:659～663
115．王晓晨，Willson A D，Guy B，等．天麻中一种抗真菌蛋白基因的克隆．植物学报，1999，41(10)：1041～1045
116．项慧新，陈桂平．植物抗逆基因分离策略．唐山师范学院学报，2002，24(5)：74～76
117．陈庆山．植物基因分离方法．中国生物工程杂志，2003，23(8)：43～46
118. Leung J, Bouvier-Durand M, MorrisPC, et al. Arabidopsis ABA response gene ABI1: feature of a calcium-modulated protein phosphatase. Science, 1994, 264: 1448～1452
119. Bryan G T, Wu K S, Farrall L, et al. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell, 2000, 12(11): 2033～2046
120. Hartmann U, HShmann S, Nettesheim K, et al. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J, 2000, 21: 351～360
121. Ambrose B A, Lerner D R, Ciceri P. et al. Molecular and genetic analysis of the Sllky1 gene reveal conservation in floral organ specification between eudicots and monocats. Mol Cell, 2000, 5: 569～579
122. Halliday K J, Hudson M, NiM, et al. pocl: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein. Proc Natl Acad Sci USA, 1999,96:5832～5837
123. Froman M A, Dush M K, Martin G R. Rapid production of full-length cdnas from rare transcripts-amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA, 1988, 85: 8998～9002
124. Ohara O, Dorit R L. One-sided polymerase chain reaction: The amplification of cDNA. Proc Natl Acad Sci USA, 1989, 86: 5673～5677
125．唐克轩，开国银，张磊，等．RACE的研究及其在植物基因克隆上的应用．复旦学报(自然科学版)，2002，41(6)：704～709
126. Suzuki, Yoshitomo-Nakagava K, Maruyana K, et al. Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library. Gene, 1997, 200, 149～156.
127．孔凡晶 马有志，陈孝，等．差异显示和RACE技术的发展与应用．生物技术通报，2003，(1)：13～16
128. De M, Linda E, Whiteman, et al. Isolation and characterisation of a cDNA clone encoding cinnamyl alcohol dehydrogenase in Eucalyptus globulus Labill. Plant Science, 1999, 143(2): 173～182
129. Reddy M K, Nair S, Singh B N, et al. Cloningand expression of a nuclear encode plastid specific 33 kDa ribonucleoprotein gene(33RNP) from pea that is light stimulated. Gene, 2001, 263(1): 179～187
130. Lingle S E, Dyear J M. Cloning and expression of sucrose synthase-1 cDNA sugarcane. Journal of Plant Physiology, 2001, 158(1): 129～131
131．田振东 柳俊，谢从华．cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA．遗传学报．2003，30(11)：996～1002
132．李德谋 肖月华，罗明，等．棉花PTS2受体基因(GhPex7)的克隆及表达分析．遗传学报，2003，30(9)：823～829
133．陈锐 高之桢，詹树萱，等．2个水稻花发育相关MADS—box基因的全序列cDNA克隆及结构分析．复旦学报(自然科学版)，2003，42(4)：570～575
134. Hsiao TC. Plant responses to water stress. Ann. Rev. Plant Physiol. 1979, 24: 519.
135．陈福明，陈顺伟．混合液法测定叶绿素含量的研究．浙江林业科技，1984，4(1)：19～23．
136．张殿忠，汪沛洪，赵会贤．测定小麦叶片游离脯氨酸含量的方法．植物生理学通讯，1990，(4)：62～65
137. Heath R L, Packer L. Photoperoxidation in isolated chloroplasts. Ⅰ. kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys, 1968, 12(5): 189～198
138. Giannoplitis C N, Ries S K. Superoxide dismutase Ⅰ: Purification and
quantitative relationship with water-soluble protein in seedling. Plant Physiol, 1977, 59: 315～318
139．刘祖祺，张石城．植物抗性生理学．北京：中国农业出版社，1994
140．福建农林大学植物生理组编．福建农林大学植物生理学实验指导．福建：福建农林大学印，2001．
141．中国科学院上海植物生理研究所，上海市植物生理学会．现代植物生理学实验指南．北京：科学出版社，1999．
142．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著(金冬雁，黎孟枫等译)．分子克隆实验指南第二版．北京：科学出版社，1992．
143．中华人民共和国国家进出口商品检验局，AOAC编译委员会．美国公职分析家协会，AOAC分析方法手册．第十五版．北京：中国科学技术出版社，1990
144．扬维霞，张建薪．小麦旗叶中甜菜碱的提取及其含量的比色测定．麦类作物，1999，19(2)：56～57
145．朱桂花，扬高文，陈晓光．应用分光光度法测定枸杞半成品饮料中的甜菜碱含量．冷饮与速冻食品工业，1998，(1)：22～24
146．J 萨姆布鲁克，D．W．拉塞尔(黄培堂，王嘉玺等译)．分子克隆实验指南．第三版 北京：科学出版社，2002
147．汪家政，范明．蛋白质技术手册．北京：科学出版社，2000
148．梁峥，赵原，李浴春，等．菠菜甜菜碱醛脱氢酶对甜菜碱醛的氧化．植物生理学报，1991，39(9)：680～686．
149．张木清．甘蔗耐旱生理基础及其化学调控．福建农林大学博士学位论文，1994．
150. Gould S J, Keller G A. Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomals proteins. Cell Biol, 1998, 107: 897～905
151．赵可夫．植物抗盐生理．北京：中国科学技术出版社，1993
152．汤章城．逆境条件下植物脯氨酸的积累及其可能的生理意义．植物生理学通讯，1984：(1)：15～21
153．韩贻仁，樊廷俊，扬晓梅，等．分子细胞生物学．第二版．北京：科学出版社，2001
154. Brouquisse R, Weigel P, Rhodes D, et al. Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma. Plant Physiol, 1989, 90: 322～329
155. Nicholas S. Plant resistance to environmental stress. Curr Opin Biotech, 1998,9:214～219
156. Brauner F, ebela M. Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues. Plant Physiology and Biochemistry, 2003, 41: 1～10
157. Ishitani M, Nakamura T, Han S Y,et al. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol,1995,27:307～315
158. Roberto V G, Lilian G S. Steady-state kinetic mechanism of the NADP~+-and NAD~+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. Biochem, 2000, 352: 675～683
159. Figueroa-Soto C G, Yalenzuela-Soto E M. Kinetic study of porcine kidney betaine aldehyde dehydrogenase. Biochem Biophys Res Commun, 2000,269(2):596～603
160．骆爱玲，赵原，李浴春，等．甜菜碱脱氢酶在细胞中的定位．植物生理学报，1995，21(2)：117～122
161. Arakawa K, Katayama M, Takabe T. Levels of betaine and betaine aldehyde dehydrogenase activity in the leaves, and etiolated leaves and roots of barley. Plant Cell Physiol, 1990,31:797～803
2．扬洪强，贾文锁，张大鹏．植物水分胁迫信号识别与转导．植物生理学通讯，2001，37(2)：149～154
3．缪颖，伍炳华．植物抗逆性的获得与信息传导．植物生理学通讯，37(1)：71～76
4．颜华，贾良辉，王根轩．植物水分胁迫诱导蛋白的研究进展．生命的化学，2002，22(2)：165～168
5．余光辉，李玲，曾福华．水分胁迫的基因表达和信号转导．亚热带植物科学，2002，31(1)：57～62
6. Shinozaki K, Yamaguchi-Shinozaki K, Mizoguchi T, et al. Molecular responses to water stress in Arabilopsis thaliana. J Plant Res, 1998, 111:345～351
7. Bray E A. Plant responses to water deficit. Trends Plant Sci, 1997, 2: 48～54
8. Meskiene I, Heft H. MAP kinase pathways: molecular plug-and-play chip for the cell. Plant Mol Biol, 2000, 42: 791～806
9. Mikami K, Katagiri T, Iuchi S, et al. A gene encoding phosphatidylinositol-4-phosphate 5-kinase is induced by water stress and abscisic acid in Arabidopsis thsliana.Plant J, 1998, 15(4): 563～568
10. Takahashi S, Katagiri T, Yamaguchi-Shinozaki, et al. An Arabidopsis gene encoding a Ca~(2+)-binding protein is induced by Abscisic Acid during dehydration. Plant and Cell Physiology, 2000, 41(7): 898～903
11. Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca~(2+)-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J, 2000, 23: 319～327
12. Uno Y, Furihata T, Abe H, et al. Arabidopsis basic leucine zipper transcription factors involved an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc Natl Acad Sci USA, 2000,97: 11632～11637
13. Liu Q, Kasuga, M, Sakuma Y, et al. Two transduction 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 Arabilopsis. Plant Cell, 1998, 10: 1391～1406
14. Nakashima K, Kiyosue T, Yamaguchi-Shinozaki K, et al. A nuclear gene erdl encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally upregulated during senescence in Arabidopsis thaliana. Plant J, 1997, 12(4): 851～861
15．刘强，张贵友，陈受宜．植物转录因子的结构与调控作用．科学通报，2000，45(14)：1465～1474
16．经艳芬，桃联安，扬李和，等．云南野生甘蔗种质血缘F_3代亲本材料抗旱性研究初报．甘蔗，2002，9(1)：19～23
17．陈能武，扬荣仲，黄久凯，等．甘蔗抗旱性鉴定方法的研究：1．甘蔗苗期抗旱性鉴定方法的筛选．四川甘蔗，1992，3(4)：5～9
18．张木清，陈如凯，余松烈．水分胁迫下蔗叶活性氧代谢的数学分析．作物学报，1996，22(6)：729～735
19．文建成，陈学宽，符菊芬，等．质膜透性与丙二醛(MDA)含量的变化评价甘蔗品种抗旱性初探．甘蔗，1998，5(3)：1～5
20．陈如凯，张木清，陆裔波．干旱胁迫对甘蔗生理影响的研究．甘蔗，1995，2(1)：1～6
21．高三基，罗俊，陈如凯，等．甘蔗品种抗旱性光合生理指标及其综合评价．作物学报，2002，28(1)：94～98
22．吴才文，陈能武，扬荣仲，等．水分胁迫及复水对甘蔗生长发育及相关生理指标的影响．甘蔗，1998，5(3)：6～12
23．徐永胜．甘蔗愈伤组织对水分胁迫的生理反应及其抗旱性．广西农学院硕士论文，1986
24. Rap C K, Asckan S. Studies on free proline association to drought resistance in sugarcane. The Sugar J, 1978, (1): 23～24
25. Rutherford R S. The assessment of profile accumulation as a mechanism of drought resistance in sugarcane. Prec S Afric Sug Tech Asseci, 1989: 136 141
26．潘世明．甘蔗抗旱性直接鉴定法的综合评价．福建甘蔗，1995，(1)：25～28
27. Inman-Bamber N G, Thompson G D. The reaction of two varieties of sugarcane to water stress. Field Crop Research, 1986, (14):15～28
28．陈少裕．甘蔗抗旱机理研究现状与展望．福建农学院学报，1991，20(1)：12～17
29．张木清，陈如凯，余松烈．水分胁迫下蔗叶多胺代谢变化及其同抗旱性的关系．植物生理学报，1996，22(3)：327～332
30. Kasuga M, Liu Q, YamaguchiS K, et al. Improving plant drought, salt tolerance by gene transfer of a single stress-inducible transporter factor. Nature Biotechnol, 1999, 17: 287～291
31. Bowler C, Slooten L, Vandenbranden S, et al. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J, 1991, 10: 1723～1732
32. Gupta A S, Heinen J L, Holaday A S, et al. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA, 1993, 90: 1629～1633
33. Dure L Ⅲ, Greenway S C, Galau G A. Developmental biochemistry of cotton seed embryogenesis and germination. ⅪⅤ. Changing mRNA populations as shown by in vitro and in vivo protein synthesis. Biochemistry, 1981, 20: 4162～4168
34. Kishor P B K, Hong Z, Miao G H, et al. Overexpression of [delta] -Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants. Plant Physiol, 1995, 108: 1387～1394
35. Holmstrom K O, Mantyla E, Welin B, et al. Drought tolerance in tobacco. Nature, 1996, 379: 683～684
36. Pilon-Smits E A H, Ebskamp M J M, Paul M J, et al. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol, 1995, 107: 125～130
37. Sheveleva E, Chmara W, Bohnert H J, et al. Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L. Plant Physiol, 1997, 115: 1211～1219
38. Capell T, Escobar C, Lui H, et al. Over-expression of the oat arginine decarboxylase cDNA in transgenic rice(Oryza sativa L.)affects normal development patterns in vltro and results in putrescine accumulation in transgenic plants. Theor Appl Genet, 1998, 97: 246～254
39. Zhu B, Su J, Chang M, et al. Overexpression of a D-pyrroline-5-carboxylate
synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice. Plant Sci, 1998, 139: 41～48
40．王自章．农杆菌介导海藻糖合酶基因遗传转化甘蔗研究．福建农林大学博士学位论文，2001
41. Falco M C. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell Report, 2000, 19(12): 301～304
42. Arencibia A, Elva, Chan, MT, et al. An efficient protocol for sugarcane (Saccharum officinarum L.)Transformation mediated by Agrobacterium tumefaciens. Transgenic Research, 1998, 7:213～222
43．田允波，葛长荣．甜菜碱在畜牧生产中的应用．内蒙古畜牧科学，1998，19(2)：10～13
44．刘建中，扬晓建，将宗勇．甜菜碱在饲料中的应用．广东饲料，1999，(5)：12～23．
45．钟伟泽．甜菜碱在饲料工业中的应用．广东饲料，1998，(5)：28～29．
46．宋丽丽，阚振荣．甜菜碱的制备、含量测定及其应用．河北林果研究，2002，17(4)：382～388
47. Weissensteiner T, Lanchbury J S. Stratergy for controlling preferential amplification and avoiding false negatives in PCR typing. Biotechniques, 1996, 21: 1102～1108
48. Henke W, Herdel K, Jung K, et al. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res, 1997,25, 3957～3958
49．梁峥，骆爱玲．甜菜碱和甜菜碱合成酶．植物生理学通讯，1995，31(1)：1～8．
50. Wyn Jones R G, Storey R. Physiology and biochemistry of drought resistance in plants. New York: Academic Press, 1981.
51. McNeil S D, Nuccio M L, Hanson A D. Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistance. Plant Physiol, 1999, 120: 945～949
52. Grumet R, Hanson A D. Genetics evidence of an osmoregulary function of glycinebetaine accumulation in barley. J Plant Physiol, 1986, 13: 353～364
53. Guy R D, Warne P G, Reid D M, et al. Glycinebetaine content of halophytes: improved analysis by liquid chromatography and interpretations of results. Physiol Plant, 1984, 61: 195～202
54．梁峥，赵原，汤岚，等．甜菜碱对呼吸酶的保护作用．植物学报．1994，36(12)：947～951．
55．侯彩霞，於新建，李荣，等．甜菜碱稳定PSⅡ放氧中心外周多肽机理．中国科学(C辑)，1998，28(4)：355～361．
56．张士功，高吉寅，宋景芝．甜菜碱对NaCl胁迫下小麦细胞保护酶活性的影响．植物学通报，1999，16(4)：429～432
57．孙文越，王辉，黄久长．外源甜菜碱对干旱胁迫下小麦幼苗膜脂过氧化作用的影响．西北植物学报，2001，21(3)：487～491
58．赵博生，衣艳君，刘家尧．外源甜菜碱对干旱／盐胁迫下的小麦幼苗生长和光合功能的改善．植物学通报，2001，18(3)：378～380
59．衣艳君，刘家尧，骆爱玲，等．转BADH基因烟草的光系统Ⅱ和呼吸酶活性的变化．植物学报，1999，41(9)：993～996
60. Nomura M, Hibino T, Takabe T, et al. Transgenically produced glycinebetaine protects ribulose-1, 5-bisphosphate carboxylase/oxygenase from inactivation in Synechococcus sp. PCC7942 under salt stress. Plant Cell Physiol, 1998, 39(4): 425～432
61．侯彩霞，汤章程．细胞相容性物质的生理功能及作用机制．植物生理学通讯，1999，35(1)：1～7．
62．许雯，孙梅好，朱亚芳，等．甘氨酸甜菜碱增强青菜抗盐作用．植物学报，2001，43(8)：809～814
63. Brouquisse R, Weigel P, Rhodes D, et al. Evidence for ferredoxin-depedent choline monooxygenase from spinach chloroplast stroma. Plant Physiol,1989, 90: 322～329.
64. Burnet M, Lafontaine P J, Hanson A D. Assay, purification, and partial characterzation of choline monooxygenase from spinach. Plant Physiol, 1995, 108: 581～588.
65. Rathinasabapathi B, Burnet M, Russell B L, et al. Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning. Proc Natl Acad Sci USA, 1997, 94(7): 3454～3458.
66. Russell B L, Rathinasabapathi B, Hanson A D. Osmotic stress induces
expression of choline monooxygenase in sugar beet and amaranth. Plant Physiol, 1998, 116(2): 859～865.
67．沈义国，杜保兴，张劲松，等．山菠菜胆碱单氧化物酶基因(CMO)的克隆与分析．生物工程学报，2001．17(1)：1～6．
68. Meng Y L, Wang Y M, Zhang B, et al. Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions. Cell Res, 2001, 11(3): 187～193.
69. Nuccio M L, Russell B L, Nolte, K D, et al. The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J, 1998,16: 487～496.
70. Nuccio M L, McNeil S D, Ziemak M J, et al. Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway. Metab Eng, 2000, 2: 300～311.
71. McNeil S D, Rhodes D, Russell B, et al. Metabolic modeling identifies key constraints on an engineered glycinebetaine synthesis pathway in tobacco. Plant Physiol, 2000, 124: 153～162.
72．李秋莉 刘大伟，高晓蓉，等．辽宁碱蓬胆碱单加氧酶基因克隆及转基因烟草的耐盐性．Acta Botanica Sinica，2003，45(2)：242～247
73. Pan S M, Moreau R A, Yu C, et al. Betaine accumulation and betaine aldehyde dehydrogenase in spinach leaves. Plant Physiol, 1981, 67: 1105～1108.
74. Weigel P, Weretilnyk E A. Betaine aldehyde oxidation by spinach chloroplasts. Plant Physiol, 1986, 82: 753～759.
75. Arakawa K, Takabe T, Sugiyama T, et al. Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. J Biochem(Tokyo), 1987,101(6):1485～1488.
76. Weretilnyk E A, Hanson A D. Betaine aldehyde dehydrogenase polymorphism in spinach: genetic and biochemical characterization. Biochem Genet, 1988, 26(1-2): 143～151.
77. Weretilnyk E A, Hanson A D. Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity. Arch Biochem Biophys, 1989, 271(1): 56～63.
78. Valenzuela-Soto E M, Munoz-Clares R A. Purification and properties of betaine aldehyde dehydrogenase extracted from detached leaves of Amarathus hypochondriacus L. subjected to water deficit. J. Plant Physiol, 1994, 143:145-152
79. Arakawa K, Mizuno K, Kishitani S, et al. Immunological studies of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol, 1992, 33(7): 833～840.
80. Liang Z, Luo A L, Tang L, et al. Betaine aldehyde dehydrogenase from spinach leaves: purification and preparation of its antibody. Chinese J Bot, 1993, 5(1): 58～64.
81. Liang Z, Li Y, Shao Y,et al. Studies on some properties of betaine aldehyde dehydrogenase from spinach. Chinese J Bot, 1993, 5(2): 154～158.
82. Ana M G, Elisa M V. Porcine kidney betaine aldehyde dehydrogenase: purification and properties. Comparative Biochemistry and Physiology(Part B 119), 1998,119: 485～491.
83. Roberto V G, Carlos M J, Guillermo M H, et al. Rapid purification and properties of betaine aldehyde dehydrogenase from pseudomonas aeruginosa. Journal of Bacteriology, 1999, 181(4): 1292～1300.
84. Hibino T, Meng Y-L, Kawamitsu Y, et al. Molecular cloning and functional characterization of two kinds of betaine aldehyde dehydrogenase in betaine accumulating mangrove Avicennia marina(Forsk.) Vierh. Plant Mol Bio, 2001,45(3):353～363.
85. Falkenberg P, Strom A R. Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli. Biochim Biophys Acta, 1990, 1034:253～259.
86. Weretilnyk E A, Hanson A D. Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. Proc Natl Acad Sci USA, 1990, 87(7): 2745～2749.
87. McCue K F, Hanson A D. Salt-inducible betaine aldehyde dehydrogenase form sugar beet: cDNA cloning and expression. Plant Mol Biol, 1992, 18: 1～11.
88．肖岗，张耕耘，刘凤华等．山菠菜甜菜碱醛脱氢酶基因研究．科学通报．1995，40(8)：741～745．
89. Legaria J, Rajsbaum R, Munoz-Clares R A, et al. Molecular characterization
of two genes encoding betaine aldehyde dehydrogenase from amaranth. Expression in leaves under short-term exposure to osmotic stress or abscisic acid. Gene, 1998,218(1-2):69～76.
90. Nakamura T, Nomura M, Mori H, et al. An isozyme of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol, 2001,42(10), 1088～1092
91. Wood A J, Saneoka H, Rhodes D, et al. Betaine aldehyde dehydrogenase in sorghum. Plant Physiol. 1996, 110(4): 1301～1308.
92. Nakamura T, Yokota S, Muramoto Y,et al. Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes. Plant J, 1997, 11(5): 1115～1120
93. Yong-Hua L, Qi Z. The sequence of Triticum aestivum Betaine Aldehyde Dehydrogenase Gene WBADH. Journal of Plant Physiology and Molecular Biology, 2002, 28(6): 495～496
94．舒卫国，艾万东，陈受宜．菠菜甜菜碱醛脱氢酶基因全序列分析．科学通报，1997，42(22)：2241～2245．
95. Brauner F, ebela M, Snégaroff J, et al. Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues. Plant Physiology and Biochemistry, 2003, 41: 1-10
96. Rathinasabapathi B, McCue K F, Gage D A, et al. Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta, 1994, 193(2): 155～162.
97．梁峥，马德钦，汤岚，等．菠菜甜菜碱醛脱氢酶基因在烟草中的表达．生物工程学报，1997，13(3)：236～240．
98．刘凤华，郭岩，谷冬梅，等．转甜菜碱醛脱氢酶基因植物的耐盐性研究．遗传学报，1997，24(1)：54～58．
99．郭岩，张莉，肖刚，等．甜菜碱醛脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究．中国科学，1997，27(2)：151～155．
100．郭北海，张艳敏，李洪杰，等．甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达．植物学报，2000，42(3)：279～283．
101．李银心，常凤启，杜立群，等．转甜菜碱醛脱氢酶基因豆瓣菜的耐盐性．植物学报，
2000，42(5)：480～484．
102．衣艳军，刘家尧，骆爱玲，等．转BABE基因烟草的光系统Ⅱ和呼吸酶活性变化．植物学报，1999，41(9)：993～996．
103. Kishitani S, Takanami T, Suzuki, M, et al. Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley. Plant Cell Environ, 2000,23:107～114
104．梁广坚．甜菜碱可以提高植物对低温的忍受力．肇庆学院学报，2003，24(2)．36～40，55
105. Diatchenko L, Lau Y F C, Campbell A P, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93:6025～6030
106. Siebert P D, Chenchik A, Kellogg DE, et al. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res, 1995, 23: 1087～1088
107. Wu G S, Saftig P, Peters C, et al. Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity. Oncogene, 1998, 16(17): 2177～2183
108. Peng L, Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerse chain reaction. Science, 1992, 257: 967～970
109. Lisitsyn N, LisitsynN, Wigler M. Cloning the differences between two complex genomes. Science, 1993, 259: 946～951
110. Hubank M, Schatz D G. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res, 1994,22:5640～5648
111. Matz M, Usman N, Shag D, et al. Ordered differential display: a simple method for systematic comparison of gene expression profiles. Nucleic Acids Res, 1997, 25(12): 2541～2542
112. Broude N E, Chandra A, Smith CL. Differential display of genome subsets containing specific interspersed repeats. Proc Nail Acad Sci USA, 1997, 94(9): 4548～4553
113. Ivanova N B, Belyavsky A V. Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting. Nucleic Acids Res, 1995,23:2954～2958
114. Prashar Y, Weissman S M. Analysis of gene expression by display of 3'end restriction fragments of cDNA. Proc Natl Acad Sci USA, 1996,93:659～663
115．王晓晨，Willson A D，Guy B，等．天麻中一种抗真菌蛋白基因的克隆．植物学报，1999，41(10)：1041～1045
116．项慧新，陈桂平．植物抗逆基因分离策略．唐山师范学院学报，2002，24(5)：74～76
117．陈庆山．植物基因分离方法．中国生物工程杂志，2003，23(8)：43～46
118. Leung J, Bouvier-Durand M, MorrisPC, et al. Arabidopsis ABA response gene ABI1: feature of a calcium-modulated protein phosphatase. Science, 1994, 264: 1448～1452
119. Bryan G T, Wu K S, Farrall L, et al. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta. Plant Cell, 2000, 12(11): 2033～2046
120. Hartmann U, HShmann S, Nettesheim K, et al. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J, 2000, 21: 351～360
121. Ambrose B A, Lerner D R, Ciceri P. et al. Molecular and genetic analysis of the Sllky1 gene reveal conservation in floral organ specification between eudicots and monocats. Mol Cell, 2000, 5: 569～579
122. Halliday K J, Hudson M, NiM, et al. pocl: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein. Proc Natl Acad Sci USA, 1999,96:5832～5837
123. Froman M A, Dush M K, Martin G R. Rapid production of full-length cdnas from rare transcripts-amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA, 1988, 85: 8998～9002
124. Ohara O, Dorit R L. One-sided polymerase chain reaction: The amplification of cDNA. Proc Natl Acad Sci USA, 1989, 86: 5673～5677
125．唐克轩，开国银，张磊，等．RACE的研究及其在植物基因克隆上的应用．复旦学报(自然科学版)，2002，41(6)：704～709
126. Suzuki, Yoshitomo-Nakagava K, Maruyana K, et al. Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library. Gene, 1997, 200, 149～156.
127．孔凡晶 马有志，陈孝，等．差异显示和RACE技术的发展与应用．生物技术通报，2003，(1)：13～16
128. De M, Linda E, Whiteman, et al. Isolation and characterisation of a cDNA clone encoding cinnamyl alcohol dehydrogenase in Eucalyptus globulus Labill. Plant Science, 1999, 143(2): 173～182
129. Reddy M K, Nair S, Singh B N, et al. Cloningand expression of a nuclear encode plastid specific 33 kDa ribonucleoprotein gene(33RNP) from pea that is light stimulated. Gene, 2001, 263(1): 179～187
130. Lingle S E, Dyear J M. Cloning and expression of sucrose synthase-1 cDNA sugarcane. Journal of Plant Physiology, 2001, 158(1): 129～131
131．田振东 柳俊，谢从华．cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA．遗传学报．2003，30(11)：996～1002
132．李德谋 肖月华，罗明，等．棉花PTS2受体基因(GhPex7)的克隆及表达分析．遗传学报，2003，30(9)：823～829
133．陈锐 高之桢，詹树萱，等．2个水稻花发育相关MADS—box基因的全序列cDNA克隆及结构分析．复旦学报(自然科学版)，2003，42(4)：570～575
134. Hsiao TC. Plant responses to water stress. Ann. Rev. Plant Physiol. 1979, 24: 519.
135．陈福明，陈顺伟．混合液法测定叶绿素含量的研究．浙江林业科技，1984，4(1)：19～23．
136．张殿忠，汪沛洪，赵会贤．测定小麦叶片游离脯氨酸含量的方法．植物生理学通讯，1990，(4)：62～65
137. Heath R L, Packer L. Photoperoxidation in isolated chloroplasts. Ⅰ. kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys, 1968, 12(5): 189～198
138. Giannoplitis C N, Ries S K. Superoxide dismutase Ⅰ: Purification and
quantitative relationship with water-soluble protein in seedling. Plant Physiol, 1977, 59: 315～318
139．刘祖祺，张石城．植物抗性生理学．北京：中国农业出版社，1994
140．福建农林大学植物生理组编．福建农林大学植物生理学实验指导．福建：福建农林大学印，2001．
141．中国科学院上海植物生理研究所，上海市植物生理学会．现代植物生理学实验指南．北京：科学出版社，1999．
142．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著(金冬雁，黎孟枫等译)．分子克隆实验指南第二版．北京：科学出版社，1992．
143．中华人民共和国国家进出口商品检验局，AOAC编译委员会．美国公职分析家协会，AOAC分析方法手册．第十五版．北京：中国科学技术出版社，1990
144．扬维霞，张建薪．小麦旗叶中甜菜碱的提取及其含量的比色测定．麦类作物，1999，19(2)：56～57
145．朱桂花，扬高文，陈晓光．应用分光光度法测定枸杞半成品饮料中的甜菜碱含量．冷饮与速冻食品工业，1998，(1)：22～24
146．J 萨姆布鲁克，D．W．拉塞尔(黄培堂，王嘉玺等译)．分子克隆实验指南．第三版 北京：科学出版社，2002
147．汪家政，范明．蛋白质技术手册．北京：科学出版社，2000
148．梁峥，赵原，李浴春，等．菠菜甜菜碱醛脱氢酶对甜菜碱醛的氧化．植物生理学报，1991，39(9)：680～686．
149．张木清．甘蔗耐旱生理基础及其化学调控．福建农林大学博士学位论文，1994．
150. Gould S J, Keller G A. Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomals proteins. Cell Biol, 1998, 107: 897～905
151．赵可夫．植物抗盐生理．北京：中国科学技术出版社，1993
152．汤章城．逆境条件下植物脯氨酸的积累及其可能的生理意义．植物生理学通讯，1984：(1)：15～21
153．韩贻仁，樊廷俊，扬晓梅，等．分子细胞生物学．第二版．北京：科学出版社，2001
154. Brouquisse R, Weigel P, Rhodes D, et al. Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma. Plant Physiol, 1989, 90: 322～329
155. Nicholas S. Plant resistance to environmental stress. Curr Opin Biotech, 1998,9:214～219
156. Brauner F, ebela M. Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues. Plant Physiology and Biochemistry, 2003, 41: 1～10
157. Ishitani M, Nakamura T, Han S Y,et al. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol,1995,27:307～315
158. Roberto V G, Lilian G S. Steady-state kinetic mechanism of the NADP~+-and NAD~+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. Biochem, 2000, 352: 675～683
159. Figueroa-Soto C G, Yalenzuela-Soto E M. Kinetic study of porcine kidney betaine aldehyde dehydrogenase. Biochem Biophys Res Commun, 2000,269(2):596～603
160．骆爱玲，赵原，李浴春，等．甜菜碱脱氢酶在细胞中的定位．植物生理学报，1995，21(2)：117～122
161. Arakawa K, Katayama M, Takabe T. Levels of betaine and betaine aldehyde dehydrogenase activity in the leaves, and etiolated leaves and roots of barley. Plant Cell Physiol, 1990,31:797～803