【作者】 张勇；

【导师】 李冠；

【作者基本信息】 新疆大学 ， 植物学， 2004， 硕士

【摘要】 近年在新疆甜瓜的生产中，微生物病害已成为影响新疆甜瓜产量和品质的重要因素。作者试图通过植物基因工程方法，利用根癌农杆菌将兔防御素基因NP-1导入新疆甜瓜中，以提高其抗病原菌危害的能力。实验中选取新疆甜瓜主栽品种：伽师瓜和皇后，建立组培再生体系。结果表明：3日龄子叶切块，均在MS＋BA1.0mg/L＋IBA 0.2 mg/L 时不定芽诱导最佳，35d有效芽诱导率分别可以达到28%和25.6％。较佳的茎段增殖培养条件为：MS+6-BA1.0mg/L+IBA0.1mg/L＋琼脂粉8g/L, pH 5.8 ,增殖系数最高达7.3 。在生根培养时，用1/2MS+0.5mg/L IBA诱导生根率较高。同时，利用根癌农杆菌介导，将广谱抗菌的NP-1基因导入伽师、皇后两个甜瓜品种。着重研究了影响甜瓜转化的外部因子：子叶外植体经3d预培养，重悬于MS液体培养基使菌液浓度达O.D600 0.6后进行侵染8～12min，再经3d共培养对转化最为有利；在侵染前进行0.2mg/L甘露醇高渗处理可提高转化率6.6%，乙酰丁香酮在甜瓜的转化中并未有显著影响。转化中卡那霉素合适筛选压力为100mg/L，转化后抑菌氨苄青霉素浓度为500mg/L有利于获取转化植株，转化率可达到12％。经卡那霉素的抗性筛选，获得转化的再生植株。分别使用PCR反应、RT-PCR反应及离体抑菌实验等手段，进一步研究了再生植株的基因整合与表达情况。经PCR和RT-PCR扩增反应的琼脂糖电泳检测，在转化植株中扩增出了特异片段，证明外源基因已整合到甜瓜基因组中，离体抑菌实验中兔防御素NP-1的转化植株能够对多种细菌表现良好的抗性，表明整合的外源基因已经得到表达。更多还原

【Abstract】 An efficient system of regeneration of XinJiang cantaloupe (Jia Shi and Huang Hou) was developed. Several regeneration protocals were analysed with particular emphasis on the regeneration efficiency. The explants excised from 3 days old seedlings cultured in MS medium supplemented with 6-BA1.0mg/L and IBA0.2mg/L，resulted in a high qualifed buds regeneration frequency (28% and 25.6% in 35days ). The shoot propagation condition is MS supplemented with 6-BA 1.0 mg/L and IBA 0.1mg/L，pH 5.8.The highest index of shoot propagation was 7.3.On the 1/2MS medium with 0.5 mg/L IBA, The high rooting initiation frequency was achieved.The cotyledons of XinJiang cantaloupes were transformed with strain of Agrobactrium tumefaciens, LBA4404, harboring expression vector pBIC-35s NP1.By selection of regenerated plantlets with Kanamycin resistance, the putative transgenic lines were believed to be obtained, In this research, the influence factors of cantaloupe transformation were discussed, the preferable protocol is 3 days cotyledon culture and resuspended bacterium, O.D600=0.6, with liquid MS before transformation. Inoculation and co-culture duration were 8~12min, 3d. And Km100mg/L, Amp500mg/L supplied to development media(MS+ BA1.0mg/L＋IBA 0.2 mg/L) is proper. During transformation, 0.2mg/L mannitol treatment is effective (efficiency increased 6.6%) but As is not so. After strict Kanamycin selection, 12 lines of putative transgenic cantaloupe were obtained. Molecular analysis of was carried to confirm the transgenic nature. The transformation of NP-1 gene was verified by PCR and RT-PCR analysis, respectively. And at the same time, the total protein was extracted to carry out the bacterium restraining experiment in vitro. The result confirmed that the transgenic cantaloupes were obtained.更多还原