Bt抗虫基因导入马铃薯的遗传转化研究
摘要
本研究通过基因工程手段, 利用农杆菌介导法将Bt基因导入马铃薯品种大西洋, 同时对马铃薯品种大西洋、地西瑞、东农303和克新4号的茎段和叶片再生体系进行优化，建立了马铃薯高效再生体系和遗传转化体系, 探讨了影响植株再生频率和遗传转化效率的因素, 确立了最佳植株再生体系和遗传转化条件，获得了转基因植株。主要研究结果如下：
1马铃薯再生体系的建立
⑴ 茎段最佳愈伤培养基为:MS+0.1 mg/l 2,4-D+1mg/l BA；最佳分化培养基为:MS+1mg/l GA3+1mg/l ZT。叶片最佳愈伤培养基为: MS+0.2mg/lNAA +5mg/l GA3 +2.25mg/l BA；最佳分化培养基为:MS+5mg/l GA3+2.25mg/lBA。
⑵ 基因型不同，在相同侵染条件下的愈伤诱导率差异较大。大西洋的愈伤率最高，茎段为68.76%，叶片达75.11%。
⑶ 以相同的农杆菌菌株介导不同类型外植体，外植体分化率不同。本试验采用茎段和叶片作为遗传转化的外植体材料，最佳外植体为叶片。
2 遗传转化影响因素探讨
(1) 茎段外植体最佳预培养时间为2d，此时抗性愈伤率最高，为56.78%。最佳共培养时间为2d，抗性愈伤率最高为50.10%。叶片外植体最佳预培养时间为2d，此时抗性愈伤率最高，为71.25%。最佳共培养时间为3d，此时抗性愈伤率最高，为70.00%。
(2) 侵染时间和菌液浓度因外植体类型而异,茎段外植体最佳侵染时间为10 min，此时抗性愈伤率最高，为54.62%，菌液浓度OD600=0.5时，抗性愈伤率最高为49.37%；叶片最佳侵染时间为10min，此时抗性愈伤形成率为63.69%，菌液浓度OD600=0.5时，抗性愈伤率最高为68.12%。
(3) 杀菌剂选用进口头孢噻肟钠。浓度为200mg/l时，可完全抑制菌的生长，且对外植体再生无影响。
(4) 抗性选择剂为卡那霉素（Kan）。在不同生长阶段，选择压力不同。在愈伤形成阶段，Kan的选择压力为50mg/l；在生根阶段，Kan的选择压力为75mg/l。
3 抗性植株的检测
试验接种转化101个叶片，获得的大西洋再生植株经PCR检测和PCR-Southern杂交检测有3株呈阳性, 转化率为2.96%, 初步证明了目的基因以整合到了马铃薯基因组中。
In this study Bt gene was introduced into Atlantic successfully with Agrobacterium
mediated method. At the same time the regeneration system of the cultivars Atlantic, Desiree, Dongnong303, Kexin number4 were optimized as well for leaf and stem explants. It established a highly efficient regeneration system of potato and transformation system were optimized. The integration of target gene into potato genome was analyzed. The factors influencing regeneration rate and transformation rate were discussed and the positive plants gained. The main results were showed as following:
1.Establishment of the regeneration system for potato
(1) Two types of explants, leaf and stem cuttings, were used in the study. For stem optimum medium for callus culture was MS+0.1 mg/l 2,4-D+1mg/l BA；the best medium for differentiation was MS+1mg/l GA3+1mg/l ZT. For leaf optimum medium for callus culture was MS+0.2mg/lNAA +5mg/l GA3+2.25mg/l BA；the best medium for differentiation was MS+5mg/l GA3 +2.25mg/lBA.
(2) Under the same infection conditions, three different genotypes of potatoes varied significantly in the percentage of culture. Atlantic have a high percentage of callus for stem with 68.76% and for leaf with 75.11%,
(3) Different explants' types interposition by the same bacterial strain, the resulting differentiation is different too. In the present studies stem and leaf are explants .The best explants was leaf.
2. Factors affecting genetic transformation
(1) For stem optimum time of pre-culture was 2d.The highest frequency was 56.78%. Optimum time of co-culture was 2d. The highest frequency was 50.10%. For leaf optimum time of pre-culture was 2d.The highest frequency was 71.25%. Optimum time of co-culture is 3d. The highest frequency was 70.00%.
（2）The infection duration and concentration of Agrobacterium varied with the explants' type. For stem, optimum infection duration was 10min, The highest frequency was 54.62%.Concentration of Agrobacterium was OD600=0.5，The highest frequency were 49.37%. For leaf optimum infection duration was 10min, The highest frequency were 63.69% .Concentration of Germ liquid was OD600=0.5，The highest frequency were 68.12%.
(3) Cefotaxime sodium was used as bactericide, which can restrain bacterium infection without any negative influence on regeneration of explants when the concentration was 200ml/l.
(4) Kanamycin was used as the selective agent. The selection pressure varied with different growth phases. During the wound-curing phase, the selection pressure of Kan was 50mg/l,
during the root-growing phase, the selection pressure of Kan is 75mg/l.
3 Test of transgenic plants
In the experiment, 101 leaf cuttings were infected by Bt gene. Three Bt gene transformed plants that is PCR tested and PCR-Southern tested positive were obtained, therefore the transformation rate was 2.96%.
Postgraduate: Su Junfeng
Major: Crop Genetics and Breeding Supervisor: Prof. Tian Xingya
Prof. Lu Cuihua
1马铃薯再生体系的建立
⑴ 茎段最佳愈伤培养基为:MS+0.1 mg/l 2,4-D+1mg/l BA；最佳分化培养基为:MS+1mg/l GA3+1mg/l ZT。叶片最佳愈伤培养基为: MS+0.2mg/lNAA +5mg/l GA3 +2.25mg/l BA；最佳分化培养基为:MS+5mg/l GA3+2.25mg/lBA。
⑵ 基因型不同，在相同侵染条件下的愈伤诱导率差异较大。大西洋的愈伤率最高，茎段为68.76%，叶片达75.11%。
⑶ 以相同的农杆菌菌株介导不同类型外植体，外植体分化率不同。本试验采用茎段和叶片作为遗传转化的外植体材料，最佳外植体为叶片。
2 遗传转化影响因素探讨
(1) 茎段外植体最佳预培养时间为2d，此时抗性愈伤率最高，为56.78%。最佳共培养时间为2d，抗性愈伤率最高为50.10%。叶片外植体最佳预培养时间为2d，此时抗性愈伤率最高，为71.25%。最佳共培养时间为3d，此时抗性愈伤率最高，为70.00%。
(2) 侵染时间和菌液浓度因外植体类型而异,茎段外植体最佳侵染时间为10 min，此时抗性愈伤率最高，为54.62%，菌液浓度OD600=0.5时，抗性愈伤率最高为49.37%；叶片最佳侵染时间为10min，此时抗性愈伤形成率为63.69%，菌液浓度OD600=0.5时，抗性愈伤率最高为68.12%。
(3) 杀菌剂选用进口头孢噻肟钠。浓度为200mg/l时，可完全抑制菌的生长，且对外植体再生无影响。
(4) 抗性选择剂为卡那霉素（Kan）。在不同生长阶段，选择压力不同。在愈伤形成阶段，Kan的选择压力为50mg/l；在生根阶段，Kan的选择压力为75mg/l。
3 抗性植株的检测
试验接种转化101个叶片，获得的大西洋再生植株经PCR检测和PCR-Southern杂交检测有3株呈阳性, 转化率为2.96%, 初步证明了目的基因以整合到了马铃薯基因组中。
In this study Bt gene was introduced into Atlantic successfully with Agrobacterium
mediated method. At the same time the regeneration system of the cultivars Atlantic, Desiree, Dongnong303, Kexin number4 were optimized as well for leaf and stem explants. It established a highly efficient regeneration system of potato and transformation system were optimized. The integration of target gene into potato genome was analyzed. The factors influencing regeneration rate and transformation rate were discussed and the positive plants gained. The main results were showed as following:
1.Establishment of the regeneration system for potato
(1) Two types of explants, leaf and stem cuttings, were used in the study. For stem optimum medium for callus culture was MS+0.1 mg/l 2,4-D+1mg/l BA；the best medium for differentiation was MS+1mg/l GA3+1mg/l ZT. For leaf optimum medium for callus culture was MS+0.2mg/lNAA +5mg/l GA3+2.25mg/l BA；the best medium for differentiation was MS+5mg/l GA3 +2.25mg/lBA.
(2) Under the same infection conditions, three different genotypes of potatoes varied significantly in the percentage of culture. Atlantic have a high percentage of callus for stem with 68.76% and for leaf with 75.11%,
(3) Different explants' types interposition by the same bacterial strain, the resulting differentiation is different too. In the present studies stem and leaf are explants .The best explants was leaf.
2. Factors affecting genetic transformation
(1) For stem optimum time of pre-culture was 2d.The highest frequency was 56.78%. Optimum time of co-culture was 2d. The highest frequency was 50.10%. For leaf optimum time of pre-culture was 2d.The highest frequency was 71.25%. Optimum time of co-culture is 3d. The highest frequency was 70.00%.
（2）The infection duration and concentration of Agrobacterium varied with the explants' type. For stem, optimum infection duration was 10min, The highest frequency was 54.62%.Concentration of Agrobacterium was OD600=0.5，The highest frequency were 49.37%. For leaf optimum infection duration was 10min, The highest frequency were 63.69% .Concentration of Germ liquid was OD600=0.5，The highest frequency were 68.12%.
(3) Cefotaxime sodium was used as bactericide, which can restrain bacterium infection without any negative influence on regeneration of explants when the concentration was 200ml/l.
(4) Kanamycin was used as the selective agent. The selection pressure varied with different growth phases. During the wound-curing phase, the selection pressure of Kan was 50mg/l,
during the root-growing phase, the selection pressure of Kan is 75mg/l.
3 Test of transgenic plants
In the experiment, 101 leaf cuttings were infected by Bt gene. Three Bt gene transformed plants that is PCR tested and PCR-Southern tested positive were obtained, therefore the transformation rate was 2.96%.
Postgraduate: Su Junfeng
Major: Crop Genetics and Breeding Supervisor: Prof. Tian Xingya
Prof. Lu Cuihua
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67 Block, M, Genotype-independent Leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumcfaciens. Theor. Appl. Genet, 1988:76: 767-774.
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71 Feher A et al. Expression of PVX coat protein gene under the control of extension –gene promoter confers virus resistance on transgenic potato plants. Plant Cell Reports 1992 ，11(1): 48-52.
72 Re G.G., D. W. Kingsbury. Paradoxical effects of sendai virus DI-RNA size on survival
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75 Jia S R et al. Genetic engineering of Chinese potato cultivars by introducing antibacterial polypeptide gene. In: Biotechnology in agriculture , Proceedings of the First Asia-Pacific Conference on Agriculture Biotechnology, Beijing ,China , 1992 , 8：20-24.
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77 Joseph, M, et al. A defective replicas gene induces resistance to cucumber mosaic virus in transgenic tobacco plants. Proc Nat Acad. Sci USA Vol, 1992, 89: 3759-3763.
78 Jongedijk E, Tigelaar H. Synergistic activity of chitinases andβ-1, 3-glucanases enhances fungal resistance in transgenic tomato plants [J]. Euphytica, 1995,85:173-180.
79 Kaniewski W et al. Field resistance of transgenic Russet Burbank potato to effects of infection by PVX and PVY biotechnology, 1990, 8:750-754.
80 Karattiger, A.F.Insect resistance in crops: A case study of Bacillus thuring- iensis (Bt) and its transfer to developing countries. ISAAA Briefs NO.2. ISAAA:I thaca, N Y.1997,PP.4-11
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83 Lin W, Anurathacs, Karabi Datta, et al. Genetic Engineering of Rice for Resistance to Sheath Blight [Ｊ].Biotechnology. 1995, 13 (7): 686-691.
84 Lamb J.W and Hay R.T. Ribozymes that cleave potato leafroll virus RNA within the coat protein and polymerase gene. Journal of General Virology，1990，71:2257-2264.
85 Montanelli C , Nascari G . Introduction of an antibacterial gene in potato using a binary vector in Agrobacterium genes, J Genet Breed .1991,45:307-316.
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90 Richter L J, Thanavala Y, Arntzen C J, et al. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Bio technol, 2000,18(11):1167-1171.
91 Rgathehouse A M,V A Hilder, and D Boulter. Plant genetic manipulation for crop protection, CAB international, 1992, 155-181.
92 Schepers, A.A Beestman, Effect of fertilizer on the susceptibility to virus infection of the potato with special reference to mature-plan resistance.proc. 12colloq.Int.potash Inst. 1976,201-210.
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