农杆菌介导ASACC基因转化苹果的研究
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摘要
本实验建立了‘新红星’苹果高效再生体系和衣杆菌介导的遗传转化体系，并采用农杆菌介导法将ASACC基因转入‘新红星’苹果，获得了携带ASACC基因的苹果新种质。研究结果如下：
1．在外植体接种培养基中添加2g/L的活性炭能有效的防止外植体褐变。
2．研究了‘新红星’苹果的高效快繁体系，比较研究发现，其扩繁效果以附加BA2.0mg/L和NAA0.1 mg/L的MS和改良C17培养基为基本培养基交替培养为佳。
3．通过研究基本培养基的类型、激素种类和浓度、暗培养时间、叶片节位、叶片放置方式等因素对‘新红星’叶片再生频率的影响，发现用第2、3、4节位的叶片，在含TDZ 0.5mg/L、NAA 0.3mg/L的MS培养基上，经过21天左右的暗培养后光照培养，‘新红星’苹果叶片再生频率达90.9％。
4．通过对叶片预培养时间、侵染时间、共培养时间、抑菌素种类和浓度、延时筛选时间、不同选择压等遗传转化影响因素的研究，建立的‘新红星’苹果遗传转化体系为：叶片黑暗预培养3天，经农杆菌侵染3分钟，在含TDZ0.5mg/L，NAA0.3mg/L的MS培养基上黑暗共培养3天，然后加250mg/L Cef黑暗维持培养3天，再进行Km12.5mg/L的选择压黑暗培养至抗性愈伤出现；转为光照条件1-2次的继代筛选，获得抗性芽后，进行扩繁。
5．实验获得了具有卡那霉素抗性的‘新红星’转化植株，PCR扩增和PCR-Southern杂交等检测结果证实，ASACC基因已整合到‘新红星’转化植株的基因组中。此外，本实验还获得了具有卡那霉素抗性的‘嘎拉’苹果转化植株，该抗性植株GUS组织化学染色结果为阳性，待进一步检测。
An effective regeneration of 'Starkrimson' and agrobacteriura-mediated transformation system were developed in this study. ASACC12 was introduced into 'Starkrimson', and a new transgenetic apple germplasm was obtained. The result were as fallow-.
1. The browning of explants was effectively inhibited when active carbon was added at 2g/L on early culture.
2. In order to found an effective propagation in vitro system for 'Starkrimson', effect of medium was compared. Result shows that shoots propagated better on alternative medium MS and modified C17 with BA3.0 mg/L and NAA0. 1 mg/L than other mediums.
3. Factors that effect percent of regeneration of 'Starkrimson' were studied, including type of basic medium, type and content of hormone, time of darkness treatment, node position of leaf, way of leaf deposition. The regeneration system which had the highest regeneration rate at 90.9% was chosen. The process was that culture leaf separated from second to fourth node was on put MS medium containing TDZ 0.5mg/L and NAA 0.3mg/L, and cultured in darkness for 21 days, then light subculture was continued.
4. Factors that influence percentage of transformation including time of preculture, dipping and coculture, type and content of antibiotic, day of delayed selection culture, intension of selection were studied. The effective transformation system of 'Starkrimson' was developed as follow: Leaves precultured in darkness for three days were dipped into agrobacterium suspension for three minutes, then were cocultured in darkness on MS medium with TDZ0.5mg/L and NAA 0.3mg/L for three days, and then cultured in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L and Cef 250mg/L. Selection culture was carried out also in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L Kml2.5mg/L till resistance callues were formed. Resistance callues were light subcultured with light for one to two times, then resistance shoots were obtained, and was propagated through continued subculture.
5. In this study, we obtained 'Starkrimson' transgenetic plants carrying ASACC gene ASACC12 identified by PCR and PCR-Southern. 'Gala' transgenetic plants detected positive to GUS dyeing were also obtained, and detection experiments were expected to be continued.
1．在外植体接种培养基中添加2g/L的活性炭能有效的防止外植体褐变。
2．研究了‘新红星’苹果的高效快繁体系，比较研究发现，其扩繁效果以附加BA2.0mg/L和NAA0.1 mg/L的MS和改良C17培养基为基本培养基交替培养为佳。
3．通过研究基本培养基的类型、激素种类和浓度、暗培养时间、叶片节位、叶片放置方式等因素对‘新红星’叶片再生频率的影响，发现用第2、3、4节位的叶片，在含TDZ 0.5mg/L、NAA 0.3mg/L的MS培养基上，经过21天左右的暗培养后光照培养，‘新红星’苹果叶片再生频率达90.9％。
4．通过对叶片预培养时间、侵染时间、共培养时间、抑菌素种类和浓度、延时筛选时间、不同选择压等遗传转化影响因素的研究，建立的‘新红星’苹果遗传转化体系为：叶片黑暗预培养3天，经农杆菌侵染3分钟，在含TDZ0.5mg/L，NAA0.3mg/L的MS培养基上黑暗共培养3天，然后加250mg/L Cef黑暗维持培养3天，再进行Km12.5mg/L的选择压黑暗培养至抗性愈伤出现；转为光照条件1-2次的继代筛选，获得抗性芽后，进行扩繁。
5．实验获得了具有卡那霉素抗性的‘新红星’转化植株，PCR扩增和PCR-Southern杂交等检测结果证实，ASACC基因已整合到‘新红星’转化植株的基因组中。此外，本实验还获得了具有卡那霉素抗性的‘嘎拉’苹果转化植株，该抗性植株GUS组织化学染色结果为阳性，待进一步检测。
An effective regeneration of 'Starkrimson' and agrobacteriura-mediated transformation system were developed in this study. ASACC12 was introduced into 'Starkrimson', and a new transgenetic apple germplasm was obtained. The result were as fallow-.
1. The browning of explants was effectively inhibited when active carbon was added at 2g/L on early culture.
2. In order to found an effective propagation in vitro system for 'Starkrimson', effect of medium was compared. Result shows that shoots propagated better on alternative medium MS and modified C17 with BA3.0 mg/L and NAA0. 1 mg/L than other mediums.
3. Factors that effect percent of regeneration of 'Starkrimson' were studied, including type of basic medium, type and content of hormone, time of darkness treatment, node position of leaf, way of leaf deposition. The regeneration system which had the highest regeneration rate at 90.9% was chosen. The process was that culture leaf separated from second to fourth node was on put MS medium containing TDZ 0.5mg/L and NAA 0.3mg/L, and cultured in darkness for 21 days, then light subculture was continued.
4. Factors that influence percentage of transformation including time of preculture, dipping and coculture, type and content of antibiotic, day of delayed selection culture, intension of selection were studied. The effective transformation system of 'Starkrimson' was developed as follow: Leaves precultured in darkness for three days were dipped into agrobacterium suspension for three minutes, then were cocultured in darkness on MS medium with TDZ0.5mg/L and NAA 0.3mg/L for three days, and then cultured in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L and Cef 250mg/L. Selection culture was carried out also in darkness on MS medium with TDZ0.5mg/L NAA0.3mg/L Kml2.5mg/L till resistance callues were formed. Resistance callues were light subcultured with light for one to two times, then resistance shoots were obtained, and was propagated through continued subculture.
5. In this study, we obtained 'Starkrimson' transgenetic plants carrying ASACC gene ASACC12 identified by PCR and PCR-Southern. 'Gala' transgenetic plants detected positive to GUS dyeing were also obtained, and detection experiments were expected to be continued.
引文
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