红树DNA转化的耐盐番茄后代细胞学观察及分子细胞遗传学鉴定
摘要
1992年以来，海南大学生物技术研究所开始用花粉管方法将红树总DNA导入普通番茄，在海滩上用海水进行耐盐性筛选，首次获得了能耐海水浇灌的耐盐番茄。本课题对获得的耐盐番茄材料进行了较系统的研究，分别从形态、细胞结构、染色体水平和分子水平等四个不同的层次考察了外源基因转入的情况及其与番茄耐性增强之间的关系，获得了与耐盐相关的一些细胞结构变异的特征，证明外源红树DNA转入了普通番茄。
本实验分两部分：第一部分、对转化番茄的形态及细胞显微结构进行观察与分析，实验表明转化番茄形态与对照相比有明显的变化，其叶面积变小，叶形收拢，这种结构可有效地减少水分的蒸发，缓解由盐胁产生的生理干旱；组织解剖结构结果显示，耐盐番茄在细胞结构上发生了明显的变异：其气孔器倾向下陷，表皮毛增多，叶脉微管组织不发达，微管束周围出现增多的贮水细胞，叶缘及其它部位表面分化出大型的多细胞分泌型结构，这些结构与转化番茄后代较强的耐盐能力有关。第二部分，通过Southern Blot和基因组原位杂交(Genomic In Situ Hybridization,GISH)，对红树转化的耐盐番茄后代分别进行了分子及细胞遗传学的鉴定，在耐盐番茄的Southern Blot中发现了特异的DNA片段，表明耐盐番茄的基因组中存在外源DNA片段；利用GISH在耐盐番茄后代中检出了外源染色体片段，同时结果还表明在不同的耐盐番茄株系中，外源DNA片段整合于番茄染色体的不同位点，其整合部位分别位于染色体的亚端部和近中部。
通过以上细胞生物学和分子生物学的研究表明，外源DNA已导入普通番茄，与其基因组整合并遗传给后代，外源红树DNA片段的渗入导致了转化植株在形态、细胞结构以及分子水平上的变异，转化番茄后代耐盐能力的增强可能与番茄基因组的这种变化有关。
Since 1992,in the Institute of Biological Science and Technology of Hainan University,the pollen tube introduction method was used to breed tomato with total DNA of Rhizophora apicularta,a salt-tolerant,seashore-grown plant. The salt-tolerant transformed tomato was firstly obtained and planted on the seashore beach and watered directly with full seawater. This study is focus on the all-around analysis of the transformed tomato,which are investigated respectively on morphological,anatomical structure of the leaves,chromosome level and molecular level. From the tests,we have obtained some structural variant features related with salt resistance and demonstrated the successful introduction of Rhizophora apicularta DNA into tomato.
This thesis contains two parts. In the first part,the morphological and anatomical structures of the leaves were investigated. As to the morphology,some difference between control and transformed tomato were found. The leaf dimensions of the transformed tomato with unextended leaves were smaller than that of control. These structure changes should help to decrease the evaporation of water effectively and release the physiological drought resulted from saline. The observation results on the anatomical structure of the leaves showed various changes in the transformed tomato:hollow stomata,well-developed epidermal hairs and aqueous tissue,unwell-developed vascular tissue and excretory structure consisted of multicell different from epidermic cells. These differently structural characteristics have higher relationship to their improvement to salt resistance. The second part is the investigation of molecular-cytogenetic characterization of the transformed tomato by means of Southern blot and genomic in situ hybri
dization (GISH). Southern blot showed the foreign DNA segments were inserted into the transformed tomato genome. Using GISH,the Rhizophora apiculota DNA sequences on tomato chromosomes were characterized. The observed cell showed signal spots and their location in different region of chromosomes in different lines.
The experimental results mentioned above demonstrated that Rhizophora apiculota DNA have been introduced into tomato and integrated with tomato genome. The obvious improvement in the salt tolerance among their progenies was most probably related with the variation of their genome.
本实验分两部分：第一部分、对转化番茄的形态及细胞显微结构进行观察与分析，实验表明转化番茄形态与对照相比有明显的变化，其叶面积变小，叶形收拢，这种结构可有效地减少水分的蒸发，缓解由盐胁产生的生理干旱；组织解剖结构结果显示，耐盐番茄在细胞结构上发生了明显的变异：其气孔器倾向下陷，表皮毛增多，叶脉微管组织不发达，微管束周围出现增多的贮水细胞，叶缘及其它部位表面分化出大型的多细胞分泌型结构，这些结构与转化番茄后代较强的耐盐能力有关。第二部分，通过Southern Blot和基因组原位杂交(Genomic In Situ Hybridization,GISH)，对红树转化的耐盐番茄后代分别进行了分子及细胞遗传学的鉴定，在耐盐番茄的Southern Blot中发现了特异的DNA片段，表明耐盐番茄的基因组中存在外源DNA片段；利用GISH在耐盐番茄后代中检出了外源染色体片段，同时结果还表明在不同的耐盐番茄株系中，外源DNA片段整合于番茄染色体的不同位点，其整合部位分别位于染色体的亚端部和近中部。
通过以上细胞生物学和分子生物学的研究表明，外源DNA已导入普通番茄，与其基因组整合并遗传给后代，外源红树DNA片段的渗入导致了转化植株在形态、细胞结构以及分子水平上的变异，转化番茄后代耐盐能力的增强可能与番茄基因组的这种变化有关。
Since 1992,in the Institute of Biological Science and Technology of Hainan University,the pollen tube introduction method was used to breed tomato with total DNA of Rhizophora apicularta,a salt-tolerant,seashore-grown plant. The salt-tolerant transformed tomato was firstly obtained and planted on the seashore beach and watered directly with full seawater. This study is focus on the all-around analysis of the transformed tomato,which are investigated respectively on morphological,anatomical structure of the leaves,chromosome level and molecular level. From the tests,we have obtained some structural variant features related with salt resistance and demonstrated the successful introduction of Rhizophora apicularta DNA into tomato.
This thesis contains two parts. In the first part,the morphological and anatomical structures of the leaves were investigated. As to the morphology,some difference between control and transformed tomato were found. The leaf dimensions of the transformed tomato with unextended leaves were smaller than that of control. These structure changes should help to decrease the evaporation of water effectively and release the physiological drought resulted from saline. The observation results on the anatomical structure of the leaves showed various changes in the transformed tomato:hollow stomata,well-developed epidermal hairs and aqueous tissue,unwell-developed vascular tissue and excretory structure consisted of multicell different from epidermic cells. These differently structural characteristics have higher relationship to their improvement to salt resistance. The second part is the investigation of molecular-cytogenetic characterization of the transformed tomato by means of Southern blot and genomic in situ hybri
dization (GISH). Southern blot showed the foreign DNA segments were inserted into the transformed tomato genome. Using GISH,the Rhizophora apiculota DNA sequences on tomato chromosomes were characterized. The observed cell showed signal spots and their location in different region of chromosomes in different lines.
The experimental results mentioned above demonstrated that Rhizophora apiculota DNA have been introduced into tomato and integrated with tomato genome. The obvious improvement in the salt tolerance among their progenies was most probably related with the variation of their genome.
引文
1．王遵亲，祝寿泉，俞仁培，中国盐渍土，北京，科学出版社，1992，250-251。
2. Volkmar K M, Hu Y, Steppuhn H., Physiological responses of plants to salinity: a review。Can J Plant Sci, 1998, 78:19-27。
3. Saiz J F, Leidi E O, Is salinity tolerance related to Na accumulation in Upland cotton seedlings? Plant and Soi, 1997, 190:67-75。
4. Glenn E P, Watson M C, O'Leary J W, Axelson R D, Comparison of salt tolerance and osmotic adjustment of low-sodium and high-sodium subspecies of the C4 halophyte, Atriplex canescens。Plant Cell Environ, 1992, 15:711-718。
5. Cheeseman J M, Mechanisms of salinity tolerance in plants. Plant Physio, 1988, 87: 547-550。
6. Smirnoff C, Thonke B, Popp M, The compatibility of D-pintol and ID-I-o-methyl-mucoinositol with malate dehydrogenase activity. Bot Acta., 1990, 103:270-273。
7．赵可夫，植物抗盐生理．北京：中国科学技术出版社，1993，9-10。
8. Delauney A J, Verma D P S, Proline biosynthesis and osmoregulation in plants. Plant J, 1993, 4:215-223。
9. Aziz A, Martin-Tanguy J, Larher F, Stress-induced changes in polyamine and tyramine levels can regulateproline accumulation in tomato leaf discs treated with sodium chloride. Physiol Plan, 1998, 104:195-202。
10. Hu C-A A, Delauney A J, Verma D P S, A bifunctional enzyme (Δ~1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. PNAS, 1992, 89:9354-9358。
11. lgarashi Y, Yoshiba Y, Sanada Y, Yamaguchi-Shinozaki K, Wada K, Shinozaki K,. Characterization of the gene for Δ~1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza L. Plant Mol Bio, 1997, 33:857-865。
12. Delauney A J, Verma D P S, A soybean gene encoding Δ~1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet, 1990, 221:299-305。
13. Kavi Kisher P B, Hong Z, Miao G-H, Hu C-AA, Verma D P S, Overexpression of Δ~1I-pyrroline-5-carboxylate synthetase increases proline production ane confer osmotolerance in the transgenic plant. Plant Physiol, 1995, 108:1387-1394。
14. Weretilmyk E A, Hanson A D, Molecular cloning of a plant betaine aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. PNAS, 1990, 87:2745-2749。
15. McCue K F, Hanson A D, Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA colning and expression. Plant Mol Biol, 1992, 18:1-11。
16. Wood A J, Saneoka H, Rhodes D, Joly R J, Goldsbrough P B, Betaine aldehyde dehydrogenase in sorghum. Molecular cloning and expression of two related genes. Plant Physiol, 1996, 110:1301-1308。
17. Ishitani M, Nakamura T, Han S Y, Takebe T, Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol, 1995, 27:307-315beet: cDNA colning and expression. Plant Mol Biol, 1992, 18:1-11。
18. Russell B L, Rathinasabapathi B, Hanson A D, Osmotic stress induces expression of choline monooxygenase in sugar beet and Anarabth. Plant Physiol., 1998, 116: 859-865。
19. Hayashi H, Alia, Mustardy L, Deshnium P, Ida M, Murata N, Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J., 1997, 12:133-142。
20．郭岩，张莉，肖岗，曹守云，谷冬梅，陈受宜，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(C辑)，1997，27 (2)：151—155。
21．张慧，董伟，周骏马，杜宝兴，谷冬梅，陈受宜，果聚糖转移酶基因的克隆及耐盐转基因烟草的培育，生物工程学报，1998，14 (2)：181—186。
22. Inf ram J, Bartels D, The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant mol Bio, 1996, 147:377-403。
23. Close T J, Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant, 1996, 97:795-803。
24. Moons A, Bauw G, Prinsen E, Van Montagu M, Straeten D V D, Molecular and physiological responses to absciaic acid and salts in roots of salt-sensitive and salt-tolerant indica rice varieties. Plant Physiol, 1995, 107:177-186。
25. Xu D P, Duan X, Wang B, Hong B, Ho D T H, Wu R, Expression of a late embryogensis abundant protein gene, HVAI, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol, 1996, 110:249-257。
26. Zhang J-S, Xie C, Li Z-Y, Chen S-Y, Expression of the plasma membrane H~+-ATPase gene in response to salt stress in a rice salt-tolerant mutant and its original variety. Theor Appl Genet, 1999, 99:1006-1011。
27. Wolf B. Frommer, Uwe Ludewig, Doris Rentsch, Taking Transgenic Plants with a Pinch of Salt, Science, 1999, 285: 1222-1223。
28. Apse M P, Aharon G S, Snedden W A, Blumwald E, Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis, Science, 1999, 285(5431): 1256-1258。
29. Nobuyuki Uozumi, Eugene J. Kim, Francisco Rubio, Takao Yamaguchi, Shoshi Muto, Akio Tsuboi, Evert P. Bakker, Tatsunosuke Nakamura, and Julian I, Schroeder, The Arabidopsis HKT1 Gene Homolog Mediates Inward Na~+ Currents in Xenopus laevis Oocytes and Na~+ Uptake in Saccharomyces cerevisiae, Plant Physiol, 2000, 122 (4): 1249-1260。
30. Hong-Xia Zhang and Eduardo Blumwald, Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit, Nature Biotechnology, 2001, 19:765-764。
31. Ana Rus, Shuji Yokoi, Altanbadrait Sharkhuu, Muppala Reddy, Byeong-ha Lee, Tracie K. Matsumoto, Hisashi Koiwa, Jian-Kang Zhu, Ray A. Bressan, and Paul M. Hasegawa, AtHKT1 is a salt tolerance determinant that controls Na~+ entry into plant roots, PNAS, 2001, 98: 14150-14155。
32. Hong-Xia Zhang, Joanna N. Hodson, John P. Williams, and Eduardo Blumwald, Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. PNAS 2001, 98: 12832-12836。
33. Rungaroon Waditee, Takashi hibion, Tatsunosuke Nakamura, Aran Incharoensakdi and Teruhiro Takabe, Overexpression of a Na~+/H~+ antiporter confers salt tolerance on a freshwater cyanobacterium, making it cpable of growth in sea water, PNAS, 2002,99(6):4109-4114。
34. Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi-Shinozaki K, Matsumoto K, Shinozaki K, A gene encoding a mitogen-activated protein kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold and water stress in Arabidopsis thaliana. PNAS, 1996, 93:756-769。
35. Xu Q, Fu H H, Gupta R, Luan S, Molecular characterization of a Tyrosine-specific protein phyosphatase encoded by a stress-responsive gene in Arabidopsis. Plant Cell 1998, 10:849-857。
36. Shinozaki K, Yamaguchi-Shinozaki K, Gene expression and signal transduction in water-stress response. Plant Physiol, 1997, 115:327-334。
37. Liu J, Zhu J-K, Acalcium sensor homlog required for plant salt tolerance. Science 1998, 280:1943-1945。
38. Yamaguchi-Shinozaki K. and Shinozaki K, A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, Iow-temperature, or high-salt stress. Plant Cell1994, 6: 251-264。
39. Yamaguchi-Shinozaki K. and Shinozaki K, Characterization of the expression of a desiccation-responsive rd29gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet, 1993, 236:331-340。
40. Kasuga M, Liu Q, Miura S, Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription facter. Nat. Biotechol. 1999, 17:287-291。
41．林栖凤，邓用川，黄薇，陈菊培，李冠一，红树DNA导入茄子获得耐盐性后代的研究，生物工程进展，2001，21 (5)：40-44。
42．林栖凤，邓用川，吴多桂，陈菊培，李冠一．耐盐辣椒分子育种，生物工程进展，1999，19 (5)：19-24。
43．林栖凤，李冠一，植物耐盐性研究进展．生物工程进展，2000，20 (2)：20-25。
44．周光宇，龚蓁蓁，王自芬，远缘杂交的分子基础——DNA 片段杂交假设的一个论证．遗传学报，1979，6(4)：405-412。
45．朱生伟，黄国存，孙敬三，外源DNA直接导入受体植物的研究进展．植物学通报，2000，17 (2)：11-16。
46．谢道昕，范云六，倪万潮，黄骏麒，苏云金芽孢杆菌杀虫晶体蛋白基因导入棉花获得转基因植物．中国科学(B辑)，1991，21(4)：367-373。
47．谢道昕，范云六，倪丕冲，苏云金芽孢杆菌杀虫基因导入中国栽培水稻品种中花11号获得转基因植物．中国科学(B辑)，1991，21(8)：830-834。
48．于元杰，尹承佾，沈法福，杨掀同，程立，应用植物分子育种技术选育棉花种质系．麦棉分子育种研究，成都，四川科学技术出版社 1995，6-13。
49．龚蓁蓁，沈慰芬，周光宇，黄骏麒，钱思颖，受粉后外源DNA导入植物技术—DNA 通过花粉管通道进入胚囊．中国科学(B辑)，1988，6：611～614。
50．周光宇，曾以申，杨晚霞，远缘杂交的分子基础．遗传学报，1980，7(2)：119-122。
51．张恒悦，于元杰，彭卫东，异种外源DNA导入棉花胚囊技术的验证．农业分子育种研究进展，北京，中国农业科技出版社，1993，129-133。
52．雷勃钧，李希臣，卢翠华，钱华，周思君，谢纬武，野生大豆外源DNA导入栽培大豆及RAPD验证．中国科学(B辑)，1994，24(6)：596-601。
53．郑红军，尹承佾，于元杰，小麦矮杆变异体的RAPD分子验证及遗传分析．麦棉分子育种研究，成都：四川科学技术出版社，1995，226-252。
54．李正理，植物制片技术[M]．北京：科学技术出版社：1991，18-91。
55．谷瑞升、蒋湘宁、郭仲琛，胡杨细胞和组织结构与其耐盐性关系的研究，植物学报，1999，41 (06)：576-579。
56．肖雯、张振霞、贾恢先，典型盐地植物小獐毛的生态学研究．西北植物学报，1997，17 (6)：1-4。
57．李广毅、高国雄、尹忠东，灰毛滨藜叶解剖结构与抗逆性研究．西北林学院学报，1995，10(1)：48-518。
58．黄振英、吴鸿、胡正海，新疆10种沙生植物旱生结构的解剖学研究．西北植物学报，1995，15 (6)：56-61。
59．陆静梅、李建东，松嫩草地五种耐盐碱植物叶表皮的解剖观察．东北师大学报(自然科学版)，1994，(03)：54-59。
60．陆静梅、张常钟、张洪芹、杨凤清，单子叶植物耐盐碱的形态解剖特征与生理适应的相关性研究．东北师大学报(自然科学版)，1994(02)：78-83。
61．黄桂玲、黄庆唱，中国红树植物的营养器官结构与生态适应(Ⅰ)．生态科学 1989，(1)：100-105。
62．王厚麟，廖绅裕．大亚湾红树及海岸植物叶片盐腺与表皮非腺毛结构．台湾海峡，2000，19 (03)：372-378。
63．林鹏，中国红树林生态系统．北京，科学出版社，1997：64-66。
64．周三，韩军丽，赵可夫，泌盐盐生植物研究进展．应用及环境生物学报，2001，7 (5)：496-501。
65. Arisz W H, Camphuis I J, Heikens H, Vantooren A J, The secretion of the salt glands of Limonium Ktze. Acta Bot Neerlandica. 1955,4(3): 321-338。
66. Ziegler H, Luttge U., Die Salzdrusen Von Limonium Vulgare Ⅱ: Die Lokalisierung des Chloride. Planta. 1967, 74:1-17。
67. Levering C A, Thomson W W., The ultrastructure of the salt gland of Spartina foliosa. Planta, 1971, 97: 183-196。
68．EG 卡特著(李正理译)，植物解剖学．北京，科学出版社，1976。
69．任昱坤，盐地硷蓬Suaedasalsa(L)Pall叶的解剖结构与生态环境关系的研究，宁夏农学院学报，1995年01期。
70．辛华，张秀芬，初庆刚，山东滨海盐生植物叶结构的比较研究．西北植物学报，1998，18(4)：584—589。
71. Pinkel D, Straume T and Gray J W, Cytogenetic analysis quantitative, high-sensitivity fluorescence hybridization. PNAS, 1986, 83: 2943-2938。
72. Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JM, Dauwerse JG, van Ommen GJ, Raap A K. High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet 1992.1:587-591。
73. Heng H Q, Squire J, Tsui L-C, High-resolution mapping of mammalian genes by in situ hybridization to free chromatin, PNAS, 1992, 89: 9509-9513。
74. Gall J G and Pardue M L, Formation and detection of RNA-DNA hybrid moleculs in cytologic preparations. PNAS, 1969, 63:378-383。
75. John H A, Birnstiel M L, Jones K W, RNA-DNA hybrids at the cytological level. Nature, 1969, 223:582-587。
76. Ried T, Baldini A, Rand T C, Ward D C. Simultaneously visualization of seven different DNA probes by in situ hybridization using fluorescence and digital imaging microscopy. PNAS, 1992.89:1388-1392。
77. Leitch I J, Leitch A R and Heslop-Harrison J S, Physical mapping of plant DNA sequences by simultaneous in sitn hybridization of two different labeled fluorescent probes.J Cell Sci, 1991, 95(3):335-333。
78. Linares C, Gonzalez J, Ferrer E and Fpminaya, The use of double fluorescence in situ of 5s rDNA genes in relation to the chromosomal lacation of 18S-5.8S-26SrDNA and a C genome specific DNA sequence in the genus Arena. Genome, 1996, 39(3): 535-542。
79. Ambros P F, Matzke M A and Matzke A J M, Detection of 1 kb unique sequence(T-DNA) in plant chromosomes by in situ hybridization. Chromosoma, 1986, 94:11-18。
80. Gustafson J P, Butler E and Mcinture C L, Physical mapping of a low-copy DNA sequence linkage groups. PNAS, 1990, 87: 1899-1902。
81．杭超，宋运淳，刘立华，玉米两个RFLP标记的原位单杂交与共杂交定位比较，遗传学报，1999，26 (1)：69-75。
82. Heiskanen M, Saitta B, Plotie A and Chu ML, Head to tail prganization of the human COL6A1 and COL6A2 genes by fiber-fish. Genomics, 1995, 29(3): 801-803。
83. Heiskanen M, Kallioniemi O and Palotie A, Fiber-fish: Experiences and refined protocol. Genet Anal. Biomol Eng, 1996, 12(5): 179-184。
84. Mukai Y and Nakahara Y, Simultaneous discri 分钟 ation of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome, 1993, 36:489-494。
85．佴文惠，刘瑞清，陈玉泽，应用染色体涂染法建立人和黑叶猴的染色体同源性，遗传学报，1999，26 (5)：474-479。
86．余定会，扬凤堂，刘瑞清，利用染色体涂染方法建立人和白眉长臂猿的比较染色体图谱，遗传学报，1997，26 (5)：417-423。
87. Wyrobek A J,J. Reprod. T oxicol. Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. Suppl, 1993., 7: 3-16。
88. Weier H U, Polikoff D, Fawcett J J, Greulich K M, Lee K H, Cram S, Chapman V M, Gray J W. Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes. Genomics, 1994, 21:641-644。
89. Wyrobek A, Lowe X, Pinkel D, Bishop J, Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization. Mol. Reprod Der, 1995, 40:259-266。
90．汪旭，T Schmid, I D Alder, 多色荧光原位杂交检测小鼠精子非整数倍体．遗传学报，1999，26 (2)：112-118。
91. Rayburn A L and Gill B S, Use of biotin-labeled probes to map specific DNA sequence on wheat chromosome, J Hered, 1985, 76:78-81。
92. Schwarzacher T, Leitch A R and Bennett M D, In situ localization of parental genomes in a wide hybrid. Ann bot, 1989, 64:315-324。
93．颜辉煌，程视宽，刘国庆，栽培稻-药用野生稻杂种F1及回交后代的基因组原位杂交鉴定．遗传学报，1999，26：157-162。
94．陈乐真，张杰，荧光原位杂交技术及其应用，细胞生物学杂志，1999，21 (4)：177-180。
95. Song Y C, Gustafson J P. The physical location of fourteen RFLP markers in rice (Oryza sativa L). Theor Appl Genet, 1995, 90: 113-119。
96. Doyle J J (1988). Isolation of plant DNA. from fresh tissue. Focus 12 (1): 13-15。
97．邓用川，曾驰，徐立新，林栖凤，李冠一，海刀豆、红树DNA的提取．海南大学学报，1994 (01)：。
98. Sambrook J, Fritsch E F, Mabiatis T. 1989, Molecular Cloning,: A Laboratory Mannual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.25-26。
99. Anamthawat-Jonsson K and Reader SM, Pre-annealing of total genomic DNA probes for simultaneous genomic in situ hybridization. Genome, 1995, 38(4): 814-816。
100．金危危，李宗芸，宁顺斌，凌定厚，李立家，宋运淳，基因枪介导质粒共转化整合模式的FISH研究，科学通报，2001，46 (15)：1281-1284。
2. Volkmar K M, Hu Y, Steppuhn H., Physiological responses of plants to salinity: a review。Can J Plant Sci, 1998, 78:19-27。
3. Saiz J F, Leidi E O, Is salinity tolerance related to Na accumulation in Upland cotton seedlings? Plant and Soi, 1997, 190:67-75。
4. Glenn E P, Watson M C, O'Leary J W, Axelson R D, Comparison of salt tolerance and osmotic adjustment of low-sodium and high-sodium subspecies of the C4 halophyte, Atriplex canescens。Plant Cell Environ, 1992, 15:711-718。
5. Cheeseman J M, Mechanisms of salinity tolerance in plants. Plant Physio, 1988, 87: 547-550。
6. Smirnoff C, Thonke B, Popp M, The compatibility of D-pintol and ID-I-o-methyl-mucoinositol with malate dehydrogenase activity. Bot Acta., 1990, 103:270-273。
7．赵可夫，植物抗盐生理．北京：中国科学技术出版社，1993，9-10。
8. Delauney A J, Verma D P S, Proline biosynthesis and osmoregulation in plants. Plant J, 1993, 4:215-223。
9. Aziz A, Martin-Tanguy J, Larher F, Stress-induced changes in polyamine and tyramine levels can regulateproline accumulation in tomato leaf discs treated with sodium chloride. Physiol Plan, 1998, 104:195-202。
10. Hu C-A A, Delauney A J, Verma D P S, A bifunctional enzyme (Δ~1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. PNAS, 1992, 89:9354-9358。
11. lgarashi Y, Yoshiba Y, Sanada Y, Yamaguchi-Shinozaki K, Wada K, Shinozaki K,. Characterization of the gene for Δ~1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza L. Plant Mol Bio, 1997, 33:857-865。
12. Delauney A J, Verma D P S, A soybean gene encoding Δ~1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet, 1990, 221:299-305。
13. Kavi Kisher P B, Hong Z, Miao G-H, Hu C-AA, Verma D P S, Overexpression of Δ~1I-pyrroline-5-carboxylate synthetase increases proline production ane confer osmotolerance in the transgenic plant. Plant Physiol, 1995, 108:1387-1394。
14. Weretilmyk E A, Hanson A D, Molecular cloning of a plant betaine aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought. PNAS, 1990, 87:2745-2749。
15. McCue K F, Hanson A D, Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA colning and expression. Plant Mol Biol, 1992, 18:1-11。
16. Wood A J, Saneoka H, Rhodes D, Joly R J, Goldsbrough P B, Betaine aldehyde dehydrogenase in sorghum. Molecular cloning and expression of two related genes. Plant Physiol, 1996, 110:1301-1308。
17. Ishitani M, Nakamura T, Han S Y, Takebe T, Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol, 1995, 27:307-315beet: cDNA colning and expression. Plant Mol Biol, 1992, 18:1-11。
18. Russell B L, Rathinasabapathi B, Hanson A D, Osmotic stress induces expression of choline monooxygenase in sugar beet and Anarabth. Plant Physiol., 1998, 116: 859-865。
19. Hayashi H, Alia, Mustardy L, Deshnium P, Ida M, Murata N, Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J., 1997, 12:133-142。
20．郭岩，张莉，肖岗，曹守云，谷冬梅，陈受宜，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(C辑)，1997，27 (2)：151—155。
21．张慧，董伟，周骏马，杜宝兴，谷冬梅，陈受宜，果聚糖转移酶基因的克隆及耐盐转基因烟草的培育，生物工程学报，1998，14 (2)：181—186。
22. Inf ram J, Bartels D, The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant mol Bio, 1996, 147:377-403。
23. Close T J, Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant, 1996, 97:795-803。
24. Moons A, Bauw G, Prinsen E, Van Montagu M, Straeten D V D, Molecular and physiological responses to absciaic acid and salts in roots of salt-sensitive and salt-tolerant indica rice varieties. Plant Physiol, 1995, 107:177-186。
25. Xu D P, Duan X, Wang B, Hong B, Ho D T H, Wu R, Expression of a late embryogensis abundant protein gene, HVAI, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol, 1996, 110:249-257。
26. Zhang J-S, Xie C, Li Z-Y, Chen S-Y, Expression of the plasma membrane H~+-ATPase gene in response to salt stress in a rice salt-tolerant mutant and its original variety. Theor Appl Genet, 1999, 99:1006-1011。
27. Wolf B. Frommer, Uwe Ludewig, Doris Rentsch, Taking Transgenic Plants with a Pinch of Salt, Science, 1999, 285: 1222-1223。
28. Apse M P, Aharon G S, Snedden W A, Blumwald E, Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis, Science, 1999, 285(5431): 1256-1258。
29. Nobuyuki Uozumi, Eugene J. Kim, Francisco Rubio, Takao Yamaguchi, Shoshi Muto, Akio Tsuboi, Evert P. Bakker, Tatsunosuke Nakamura, and Julian I, Schroeder, The Arabidopsis HKT1 Gene Homolog Mediates Inward Na~+ Currents in Xenopus laevis Oocytes and Na~+ Uptake in Saccharomyces cerevisiae, Plant Physiol, 2000, 122 (4): 1249-1260。
30. Hong-Xia Zhang and Eduardo Blumwald, Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit, Nature Biotechnology, 2001, 19:765-764。
31. Ana Rus, Shuji Yokoi, Altanbadrait Sharkhuu, Muppala Reddy, Byeong-ha Lee, Tracie K. Matsumoto, Hisashi Koiwa, Jian-Kang Zhu, Ray A. Bressan, and Paul M. Hasegawa, AtHKT1 is a salt tolerance determinant that controls Na~+ entry into plant roots, PNAS, 2001, 98: 14150-14155。
32. Hong-Xia Zhang, Joanna N. Hodson, John P. Williams, and Eduardo Blumwald, Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. PNAS 2001, 98: 12832-12836。
33. Rungaroon Waditee, Takashi hibion, Tatsunosuke Nakamura, Aran Incharoensakdi and Teruhiro Takabe, Overexpression of a Na~+/H~+ antiporter confers salt tolerance on a freshwater cyanobacterium, making it cpable of growth in sea water, PNAS, 2002,99(6):4109-4114。
34. Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi-Shinozaki K, Matsumoto K, Shinozaki K, A gene encoding a mitogen-activated protein kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold and water stress in Arabidopsis thaliana. PNAS, 1996, 93:756-769。
35. Xu Q, Fu H H, Gupta R, Luan S, Molecular characterization of a Tyrosine-specific protein phyosphatase encoded by a stress-responsive gene in Arabidopsis. Plant Cell 1998, 10:849-857。
36. Shinozaki K, Yamaguchi-Shinozaki K, Gene expression and signal transduction in water-stress response. Plant Physiol, 1997, 115:327-334。
37. Liu J, Zhu J-K, Acalcium sensor homlog required for plant salt tolerance. Science 1998, 280:1943-1945。
38. Yamaguchi-Shinozaki K. and Shinozaki K, A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, Iow-temperature, or high-salt stress. Plant Cell1994, 6: 251-264。
39. Yamaguchi-Shinozaki K. and Shinozaki K, Characterization of the expression of a desiccation-responsive rd29gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet, 1993, 236:331-340。
40. Kasuga M, Liu Q, Miura S, Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription facter. Nat. Biotechol. 1999, 17:287-291。
41．林栖凤，邓用川，黄薇，陈菊培，李冠一，红树DNA导入茄子获得耐盐性后代的研究，生物工程进展，2001，21 (5)：40-44。
42．林栖凤，邓用川，吴多桂，陈菊培，李冠一．耐盐辣椒分子育种，生物工程进展，1999，19 (5)：19-24。
43．林栖凤，李冠一，植物耐盐性研究进展．生物工程进展，2000，20 (2)：20-25。
44．周光宇，龚蓁蓁，王自芬，远缘杂交的分子基础——DNA 片段杂交假设的一个论证．遗传学报，1979，6(4)：405-412。
45．朱生伟，黄国存，孙敬三，外源DNA直接导入受体植物的研究进展．植物学通报，2000，17 (2)：11-16。
46．谢道昕，范云六，倪万潮，黄骏麒，苏云金芽孢杆菌杀虫晶体蛋白基因导入棉花获得转基因植物．中国科学(B辑)，1991，21(4)：367-373。
47．谢道昕，范云六，倪丕冲，苏云金芽孢杆菌杀虫基因导入中国栽培水稻品种中花11号获得转基因植物．中国科学(B辑)，1991，21(8)：830-834。
48．于元杰，尹承佾，沈法福，杨掀同，程立，应用植物分子育种技术选育棉花种质系．麦棉分子育种研究，成都，四川科学技术出版社 1995，6-13。
49．龚蓁蓁，沈慰芬，周光宇，黄骏麒，钱思颖，受粉后外源DNA导入植物技术—DNA 通过花粉管通道进入胚囊．中国科学(B辑)，1988，6：611～614。
50．周光宇，曾以申，杨晚霞，远缘杂交的分子基础．遗传学报，1980，7(2)：119-122。
51．张恒悦，于元杰，彭卫东，异种外源DNA导入棉花胚囊技术的验证．农业分子育种研究进展，北京，中国农业科技出版社，1993，129-133。
52．雷勃钧，李希臣，卢翠华，钱华，周思君，谢纬武，野生大豆外源DNA导入栽培大豆及RAPD验证．中国科学(B辑)，1994，24(6)：596-601。
53．郑红军，尹承佾，于元杰，小麦矮杆变异体的RAPD分子验证及遗传分析．麦棉分子育种研究，成都：四川科学技术出版社，1995，226-252。
54．李正理，植物制片技术[M]．北京：科学技术出版社：1991，18-91。
55．谷瑞升、蒋湘宁、郭仲琛，胡杨细胞和组织结构与其耐盐性关系的研究，植物学报，1999，41 (06)：576-579。
56．肖雯、张振霞、贾恢先，典型盐地植物小獐毛的生态学研究．西北植物学报，1997，17 (6)：1-4。
57．李广毅、高国雄、尹忠东，灰毛滨藜叶解剖结构与抗逆性研究．西北林学院学报，1995，10(1)：48-518。
58．黄振英、吴鸿、胡正海，新疆10种沙生植物旱生结构的解剖学研究．西北植物学报，1995，15 (6)：56-61。
59．陆静梅、李建东，松嫩草地五种耐盐碱植物叶表皮的解剖观察．东北师大学报(自然科学版)，1994，(03)：54-59。
60．陆静梅、张常钟、张洪芹、杨凤清，单子叶植物耐盐碱的形态解剖特征与生理适应的相关性研究．东北师大学报(自然科学版)，1994(02)：78-83。
61．黄桂玲、黄庆唱，中国红树植物的营养器官结构与生态适应(Ⅰ)．生态科学 1989，(1)：100-105。
62．王厚麟，廖绅裕．大亚湾红树及海岸植物叶片盐腺与表皮非腺毛结构．台湾海峡，2000，19 (03)：372-378。
63．林鹏，中国红树林生态系统．北京，科学出版社，1997：64-66。
64．周三，韩军丽，赵可夫，泌盐盐生植物研究进展．应用及环境生物学报，2001，7 (5)：496-501。
65. Arisz W H, Camphuis I J, Heikens H, Vantooren A J, The secretion of the salt glands of Limonium Ktze. Acta Bot Neerlandica. 1955,4(3): 321-338。
66. Ziegler H, Luttge U., Die Salzdrusen Von Limonium Vulgare Ⅱ: Die Lokalisierung des Chloride. Planta. 1967, 74:1-17。
67. Levering C A, Thomson W W., The ultrastructure of the salt gland of Spartina foliosa. Planta, 1971, 97: 183-196。
68．EG 卡特著(李正理译)，植物解剖学．北京，科学出版社，1976。
69．任昱坤，盐地硷蓬Suaedasalsa(L)Pall叶的解剖结构与生态环境关系的研究，宁夏农学院学报，1995年01期。
70．辛华，张秀芬，初庆刚，山东滨海盐生植物叶结构的比较研究．西北植物学报，1998，18(4)：584—589。
71. Pinkel D, Straume T and Gray J W, Cytogenetic analysis quantitative, high-sensitivity fluorescence hybridization. PNAS, 1986, 83: 2943-2938。
72. Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JM, Dauwerse JG, van Ommen GJ, Raap A K. High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet 1992.1:587-591。
73. Heng H Q, Squire J, Tsui L-C, High-resolution mapping of mammalian genes by in situ hybridization to free chromatin, PNAS, 1992, 89: 9509-9513。
74. Gall J G and Pardue M L, Formation and detection of RNA-DNA hybrid moleculs in cytologic preparations. PNAS, 1969, 63:378-383。
75. John H A, Birnstiel M L, Jones K W, RNA-DNA hybrids at the cytological level. Nature, 1969, 223:582-587。
76. Ried T, Baldini A, Rand T C, Ward D C. Simultaneously visualization of seven different DNA probes by in situ hybridization using fluorescence and digital imaging microscopy. PNAS, 1992.89:1388-1392。
77. Leitch I J, Leitch A R and Heslop-Harrison J S, Physical mapping of plant DNA sequences by simultaneous in sitn hybridization of two different labeled fluorescent probes.J Cell Sci, 1991, 95(3):335-333。
78. Linares C, Gonzalez J, Ferrer E and Fpminaya, The use of double fluorescence in situ of 5s rDNA genes in relation to the chromosomal lacation of 18S-5.8S-26SrDNA and a C genome specific DNA sequence in the genus Arena. Genome, 1996, 39(3): 535-542。
79. Ambros P F, Matzke M A and Matzke A J M, Detection of 1 kb unique sequence(T-DNA) in plant chromosomes by in situ hybridization. Chromosoma, 1986, 94:11-18。
80. Gustafson J P, Butler E and Mcinture C L, Physical mapping of a low-copy DNA sequence linkage groups. PNAS, 1990, 87: 1899-1902。
81．杭超，宋运淳，刘立华，玉米两个RFLP标记的原位单杂交与共杂交定位比较，遗传学报，1999，26 (1)：69-75。
82. Heiskanen M, Saitta B, Plotie A and Chu ML, Head to tail prganization of the human COL6A1 and COL6A2 genes by fiber-fish. Genomics, 1995, 29(3): 801-803。
83. Heiskanen M, Kallioniemi O and Palotie A, Fiber-fish: Experiences and refined protocol. Genet Anal. Biomol Eng, 1996, 12(5): 179-184。
84. Mukai Y and Nakahara Y, Simultaneous discri 分钟 ation of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome, 1993, 36:489-494。
85．佴文惠，刘瑞清，陈玉泽，应用染色体涂染法建立人和黑叶猴的染色体同源性，遗传学报，1999，26 (5)：474-479。
86．余定会，扬凤堂，刘瑞清，利用染色体涂染方法建立人和白眉长臂猿的比较染色体图谱，遗传学报，1997，26 (5)：417-423。
87. Wyrobek A J,J. Reprod. T oxicol. Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. Suppl, 1993., 7: 3-16。
88. Weier H U, Polikoff D, Fawcett J J, Greulich K M, Lee K H, Cram S, Chapman V M, Gray J W. Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes. Genomics, 1994, 21:641-644。
89. Wyrobek A, Lowe X, Pinkel D, Bishop J, Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization. Mol. Reprod Der, 1995, 40:259-266。
90．汪旭，T Schmid, I D Alder, 多色荧光原位杂交检测小鼠精子非整数倍体．遗传学报，1999，26 (2)：112-118。
91. Rayburn A L and Gill B S, Use of biotin-labeled probes to map specific DNA sequence on wheat chromosome, J Hered, 1985, 76:78-81。
92. Schwarzacher T, Leitch A R and Bennett M D, In situ localization of parental genomes in a wide hybrid. Ann bot, 1989, 64:315-324。
93．颜辉煌，程视宽，刘国庆，栽培稻-药用野生稻杂种F1及回交后代的基因组原位杂交鉴定．遗传学报，1999，26：157-162。
94．陈乐真，张杰，荧光原位杂交技术及其应用，细胞生物学杂志，1999，21 (4)：177-180。
95. Song Y C, Gustafson J P. The physical location of fourteen RFLP markers in rice (Oryza sativa L). Theor Appl Genet, 1995, 90: 113-119。
96. Doyle J J (1988). Isolation of plant DNA. from fresh tissue. Focus 12 (1): 13-15。
97．邓用川，曾驰，徐立新，林栖凤，李冠一，海刀豆、红树DNA的提取．海南大学学报，1994 (01)：。
98. Sambrook J, Fritsch E F, Mabiatis T. 1989, Molecular Cloning,: A Laboratory Mannual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.25-26。
99. Anamthawat-Jonsson K and Reader SM, Pre-annealing of total genomic DNA probes for simultaneous genomic in situ hybridization. Genome, 1995, 38(4): 814-816。
100．金危危，李宗芸，宁顺斌，凌定厚，李立家，宋运淳，基因枪介导质粒共转化整合模式的FISH研究，科学通报，2001，46 (15)：1281-1284。