水稻蜡质基因启动子在玉米中的应用研究及bkt基因玉米表达载体的构建
摘要
水稻蜡质基因(Waxy)编码结合在淀粉粒上的淀粉合成酶,它控制花粉、胚乳与胚囊中直链淀粉的合成,是一种组织与发育阶段专一性表达的基因。本研究工作通过基因枪转化技术连接报道基因GUS 的水稻蜡质基因启动子载体导入玉米愈伤组织,取转化后48h 玉米幼胚,GUS 染色后作石蜡切片,肉眼即可见大面积蓝色斑块,转化后45d 玉米愈伤组织GUS 染色后作石蜡切片仍然可看到蓝色斑块,未转化玉米均未见蓝色斑块。显示水稻Wx 基因启动子可以驱动GUS 基因在发育的玉米幼胚和愈伤组织中表达。
CaMV35S 启动子是植物中常用到的组织非特异性启动子,水稻蜡质基因启动子是胚乳特异性启动子。将β-胡萝卜素酮化酶基因(bkt)分别连入这两个启动子下,引入玉米后,一方面可以比较启动子不同造成转基因玉米类胡萝卜素的各个组分的差异,另一方面也可论证玉米种子在水稻蜡质基因启动子作用下产虾青素的能力,开发其多种用途。
Tissue-specific promoter can drive foreign genes’expression in plant’s specific tissue and specific time.Rice wary gene encodes the starch synthesizing enzyme combining to starch,it controls the synthesizing of amylase in the farina、endosperm and megaspore.The rice wary gene promoter and GUS gene were linked and transformed to maize type II callus. Many blue spots were observed in the transgenic maize callus by histochemical staining, however there was no in the comparision.45 days later ,the blue sports can still be seen by histochemical staining.The results suggest that rice wary gene promoter an drive foreign genes’expression in maize endosperm. Furthermore, foreign genes’expression in the different part of maize will be checked to see if it is exist and if it is strong or unconspicuous by steady heredity.
Early nptII gene was used for screening of the transgenic maize.Now it is found that there is natural resistive in the maize tissue to the nptII gene.So nptII gene is gradually substituted by hpt and pat gene. It is proved that maize calli was sensitive to Hygromycin, 5μg/ml Hygromycin will obviously restrain the growth of maize callus.With the increasing of Hygromycin concentration,the death of maize calli increases.When the Hygromycin concentration is 50μg/ml, all the maize calli will die.The half death is 20μg/ml.
There is much Zeaxanthin in the maize but not find Astaxanthin.It is known from the synthesizing way of isoprenoids that Zeaxanthin being catalyzed by bkt gene may be made into Astaxanthin. This research bkt gene was linked with CaMV35S promoter or rice waxy gene promoter
CaMV35S 启动子是植物中常用到的组织非特异性启动子,水稻蜡质基因启动子是胚乳特异性启动子。将β-胡萝卜素酮化酶基因(bkt)分别连入这两个启动子下,引入玉米后,一方面可以比较启动子不同造成转基因玉米类胡萝卜素的各个组分的差异,另一方面也可论证玉米种子在水稻蜡质基因启动子作用下产虾青素的能力,开发其多种用途。
Tissue-specific promoter can drive foreign genes’expression in plant’s specific tissue and specific time.Rice wary gene encodes the starch synthesizing enzyme combining to starch,it controls the synthesizing of amylase in the farina、endosperm and megaspore.The rice wary gene promoter and GUS gene were linked and transformed to maize type II callus. Many blue spots were observed in the transgenic maize callus by histochemical staining, however there was no in the comparision.45 days later ,the blue sports can still be seen by histochemical staining.The results suggest that rice wary gene promoter an drive foreign genes’expression in maize endosperm. Furthermore, foreign genes’expression in the different part of maize will be checked to see if it is exist and if it is strong or unconspicuous by steady heredity.
Early nptII gene was used for screening of the transgenic maize.Now it is found that there is natural resistive in the maize tissue to the nptII gene.So nptII gene is gradually substituted by hpt and pat gene. It is proved that maize calli was sensitive to Hygromycin, 5μg/ml Hygromycin will obviously restrain the growth of maize callus.With the increasing of Hygromycin concentration,the death of maize calli increases.When the Hygromycin concentration is 50μg/ml, all the maize calli will die.The half death is 20μg/ml.
There is much Zeaxanthin in the maize but not find Astaxanthin.It is known from the synthesizing way of isoprenoids that Zeaxanthin being catalyzed by bkt gene may be made into Astaxanthin. This research bkt gene was linked with CaMV35S promoter or rice waxy gene promoter
引文
1. 丁群星,谢友菊,戴景瑞,用子房注射法将毒蛋白基因导入玉米的研究,中国科学B 辑,1993,23:707
2. 王国英,杜天兵,张宏等,用基因枪将毒蛋白基因转入玉米及转基因植株的再生,中国科学B 辑,1995,25:71
3. 贾士荣,T-DNA 转移机理,植物生理学通讯,1994,30:306
4. 颜昌敬主编,农作物组织培养,上海科学技术出版社,1991,300
5. 马红梅,朱小平,楼小明等,籼稻蜡质基因5’上游区与GUS 基因编码区融合基因的构建和在水稻中的瞬间表达,遗传学报,1995,22(5):353-360
6. 余云舟,王罡,金宁一等,农杆菌介导优良玉米自交系II 型胚性愈伤组织遗传转化体系的建立,玉米科学,2003,11(3):28-30,33
7. 黄璐,卫志明,玉米的遗传转化,植物生理学通讯,1997,33(3):226-232
8. Sangtong V, Moran DL, Chikwamba R, et al. Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize[J]. Theor Appl Genet,2002,105:937-945
9. 张海利,吕淑霞,田颍川,韧皮部特异性启动子研究概述,中国生物工程杂志,2003,23(11):11-15
10. Caimi PG, McCole ML, Klein TM, et al. Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus
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46. 侯艳华,徐仲,苍晶等,玉米自交系愈伤组织的诱导及再生,吉林农业大学学报,2002,24(6):7-10
47. 曹毅,陈英,蒋彦等,利用电击法转化玉米胚性愈伤组织获得GUS瞬间表达的研究,四川大学学报(自然科学版),2001,38(6):913-918
48. 刘大文,王守才,谢友菊等,转Zm13-Barnase 基因玉米的获得及其花粉育性研究,植物学报,2000,42(6):611-615
49. 张艳贞,胡汉桥等,农杆菌介导将Bt 杀虫蛋白基因导入优良玉米自交系的研究,遗传,2002,24(1):35-39
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2. 王国英,杜天兵,张宏等,用基因枪将毒蛋白基因转入玉米及转基因植株的再生,中国科学B 辑,1995,25:71
3. 贾士荣,T-DNA 转移机理,植物生理学通讯,1994,30:306
4. 颜昌敬主编,农作物组织培养,上海科学技术出版社,1991,300
5. 马红梅,朱小平,楼小明等,籼稻蜡质基因5’上游区与GUS 基因编码区融合基因的构建和在水稻中的瞬间表达,遗传学报,1995,22(5):353-360
6. 余云舟,王罡,金宁一等,农杆菌介导优良玉米自交系II 型胚性愈伤组织遗传转化体系的建立,玉米科学,2003,11(3):28-30,33
7. 黄璐,卫志明,玉米的遗传转化,植物生理学通讯,1997,33(3):226-232
8. Sangtong V, Moran DL, Chikwamba R, et al. Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize[J]. Theor Appl Genet,2002,105:937-945
9. 张海利,吕淑霞,田颍川,韧皮部特异性启动子研究概述,中国生物工程杂志,2003,23(11):11-15
10. Caimi PG, McCole ML, Klein TM, et al. Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus
11. 白建荣, 侯变英, 郭秀荣. 分子标记在植物育种中的应用山西农业 科学. 1999, 27(1):86-89
12. 曹小风, 杨美珠, 朱玉贤. 马铃薯病毒外壳蛋白基因在转基因马铃薯中的表达植物学报. 1996,38(3):417-420
13. 丁群星. 用基因枪法将Bt 毒蛋白基因导入玉米的研究中国科学(B辑). 1993, 23(7):707-713
14. 董延瑜. 植物基因转移研究进展湖南农学院学报1994,20(6):573-584
15. 董云洲, 段胜军, 赵连元. 用基因枪转化花粉获得转基因谷子和玉米中国农业科学.1999,31(2):9-13
16. 冯勤, 杨美珠, 郑学勤. 根癌土壤农杆菌介导的咖啡转化生物工程学报.1992, 8
17. Amstrong C. L., Green C E. Establishment and maintenance of friable, embryogenic maize callus and involvement of L-proline [J]. Planta. 1985,164:207-214
18. Amstrong C.L., Peterson W.L., Buchholz W.G. Factors effecting PEG-mediated Stable transformation of maize protoplasts [J]. Plant Cell Rep.1990, 9:335
19. Antonelli N.M., Stabler J. Chemical methods for direct gene transfer to protoplasts. Recovery of Stable transformation after direct gene transfer using polyethylene glycol (PEG) [J]. Maize Genetics Cooperation News Letter. 1989, 63:23
20. Antonelli N. M., Satdle J. Genomic DNA can be used with Cation methods for highly efficient transformation of maize protoplasts [J]. Theor APPL Genet. 1990, 80:395
21. Bonolt.A.D., Eggermont.K., Druart P. Agrabaterium-mediateed transformation of apple:an assessment of factors affecting gene transfer
efficiency during early transformation steps Plant Cell Report.
1994,13:587-593
22. Brettschneider R., Becker D., Lorz H. Efficient transformation of scutellar tissue of immature maize embryos [J]. Theor Appl Genet. 1997, 94:737-748
23. Brich R.G. Plant Transformation Problems and Strategies for Practical Appliciation. Annu Rev. Plant Mol. Biol. 1997, 48:297-326
24. Hilder V. A., Gatehouse A. M. R., Sheerman S. E. Novel mechanism of insect resistance engineered into tobacco. Nature. 1987,300 (12) 160-165
25. Hilder V. A., Powell K.S., Gatehouse A. M. R. Gene-transfer and plant regeneration techniques. Transgenic Research. 1995, 4:18-25
26. Hodges T.K., Kamo K.K., Imbrie C. W. , Becwar M.R. Genotype specificity of somatic embryogenesis and regeneration in maize. Bio/Technology. 1986, 4:219-223
27. Ishida Y., Saito H., Ohta S., Hiei Y., Komari T., Kumashiro T. High efficiency transformation of maize(Zea mays L)mediated by Agrobacterium tumefaciens. Nature Biotechnology. 1996, 14:745-750
28. Jefferson R.A., Kavanagh T. A., Bevan M. W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J. 1987, 6:3901-3907
29. Jefferson R.A. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 1987,5:387-405
30. Kaeppler H. F. Silicon carbide fiber-mediated stable transformation of plant cells [J]. Theor Appl Genet. 1992, 84:560-566
31. Kamo K.K., Hodges T.K. Eatablishment and characterization of long-term embryogenic maize callus and ceti-suspension ccultures[J]. Plant Sci. 1986,45:111-117
32. Kiyosue T., Yamaguchi-Shinozaki K., Shinozaki K., Kamada H., Harada H. cDNA cloing of EP40,an embryogenic cell protein in carrot, and its expression during somatic and zygotic embryogenesis [J]. Plant Molecular Biology. 1993, 21:1053-1068
33. Klein T.M., Kornstein L., Sanford J.C., Fromm M.E. Genetic transformation of maize cells by particle bombardment. Plant Physiol. 1989,91:440-444
34. Klein T.M., Gardziel T., Fromm M.E. Factors influencing gene delivery into zea mays cells by high-velocity microprojectiles. Bio/Technology. 1988,6:559-563
35. Koziel M.G., Beland C., Bowman C., Carozzi N.B., Crenshaw R., Crossland L., Dawson J., Desai N., Hill M., Kadwell S., Launis K., Lewis K., Maddox D., Mcpherson K., Meghji M.R., Merlin E., Rhodes R., Warren G.W., Wright M., Evola S.V. Field performance of elite transgenic maize plants expressing an in secticidal protein derived from Bacillus thurigiensis . Bio/technology. 1993,14:194-200
36. Krattiger A. F. Insect Resistance in crops: A Case Study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs No. 2. ISAAA: I thaca, N Y. 1997, 4-11
37. Laursen C.M., Krzyzek R.A., Flick C.E., Production of fertile transgenic maize by electroporation of suspension culture cells. Plant Mol Biol. 1999,4: 24:51
38. Leemans J., Reynaerts A., Hofte H. In: New Direcions in biological Control: Alternative for Suppressing Agricultrual Pests and diseases. Alan R Liss. 1990,573-581
39. Liener I. E. in: The Lectins(edi by I e Liener) Academic press. Sandiego. 1980,527-552
40. 张锐,郭三堆,植物抗虫基因工程研究进展,生物技术通报,2001,2:8-12
41. 付荣昭,植物遗传转化手册,中国科学技术出版社,1994
42. 李国胜,杨爱芳,张举仁等,玉米愈伤组织的遗传转化及抗除草剂植株再生,科学通报,2000,45(20):2181-2184
43. 吴敏生,黄健秋,卫志明等,玉米幼胚高效再生体系的建立,植物生理学报,2001,27(6):489-494
44. 韩双燕,郭勇等,农杆菌及基因枪在玉米遗传转化中的应用,广西植物,2002,22(3):259-263
45. 良越,孙晓丽,韦小敏等,玉米胚性愈伤组织诱导和植株再生研究,河南农业大学学报,2002,36(2):101-105
46. 侯艳华,徐仲,苍晶等,玉米自交系愈伤组织的诱导及再生,吉林农业大学学报,2002,24(6):7-10
47. 曹毅,陈英,蒋彦等,利用电击法转化玉米胚性愈伤组织获得GUS瞬间表达的研究,四川大学学报(自然科学版),2001,38(6):913-918
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