摘要
黄瓜是人们喜食的蔬菜作物之一，在我国蔬菜周年供应上占有重要地位，但易受多种真菌病害的危害而使产量降低，品质下降。传统的育种方法周期长、耗资大、效率低、受抗性基因资源匮乏的限制、还易出现抗性退化的现象，用现有品种资源进行有性杂交育种很难尽快得到具有抗性的优良品种。因此，本研究通过基因工程手段，利用农杆菌介导法将菜豆几丁质酶基因导入黄瓜，建立了黄瓜高效的植株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主要因素，确立了最佳的植株再生和遗传转化条件，获得了阳性转基因植株。主要研究结果如下：
1外植体和6-BA浓度对器官发生的影响 子叶节是最佳的外植体；最适宜的芽诱导培养基为MS+6-BA0.5mg／L。
2黄瓜遗传转化的最适宜条件 子叶节预培养1～2天，在含有6-BA0.5mg／L、乙酰丁香酮(AS)100μmol／L、pH5.2的MS培养基上进行培养，遗传转化效率最高。最佳的选择压力为卡那霉素30mg／L。
3转基因植株的检测 用几丁质酶两端序列合成的引物对To代再生植株进行PCR检测，结果“长春密刺”的78株再生植株中，有30株为阳性，占再生植株的38.7％。PCR检测结果可初步证明目的基因已整合到黄瓜基因组中。
Cucumber is one kind of favorite vegetables and plays an important role in vegetable supply, but it was suffered from many kinds of Fungi easily, which can decrease its yield and deteriorate its quality. Traditional method of breeding has many disadvantages, such as long period, huge investment and low efficiency. It is also restricted to the shortage of resistant genes. There is devolution of resistance in resistant cultivar. Cucumber exist serious hamper of sexual hybridization. So it is difficult to obtain the superior cultivar with high resistance. This study established a highly efficient regeneration system of cucumber and then transfer the fungi-resistant gene (bean chitinase gene) into cucumber, which mediated by Agrobacterium tumefaciens. This has not been studied in previous reports in China. The integration of target gene into cucumber genome was analyzed. The factors influencing regeneration rate and transformation rate were discussed.
The results was summarized as follows:
1 Effect of explants and concentration of 6-BA on organgenesis The best explants was cotyledonary nodes; The optimum shoot inducing medium is MS+6-BA0.5mg/L.
2 The optimum procedure in cucumber transformation as follows: The cotyledonary nodes was pre-cultured for 1~2 days, co-cultivated in medium including 6-BA 0.5mg/L and AS l00$mol/L, pH 5.2. The optimum concentration of kanamycin used in resistant shoots selection was 30mg/L.
3 Detection of transforming regenerated cucumber 30 of 78 regenerated plants of "Chang Chun Mi Ci" were positive and the percentage is 38.7%. The results of PCR demonstrated the integration of target gene into cucumber genome primarily.
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