转几丁质酶和核糖体失活蛋白双价基因大豆的获得与抗病性鉴定

以东北地区4个主栽大豆品种为试材，通过农杆菌介导法，将2个抗真菌病基因，即几丁质酶基因和核糖体失活蛋白基因构建在同一植物表达载体上，对受体材料进行遗传转化，获得了经Southern检测同时整合双价基因的T₀代转基因大豆植株，转化频率为0.5%。将T₀代转基因大豆植株扩繁至T₂代，并进行了大豆疫霉根腐病和大豆灰斑病的抗性检测，4个株系的抗性均比对照有明显提高。分子生物学验证及RT-PCR检测表明2个外源基因在4个株系中均进行了转录表达。

Obtainment of Transgenic Soybean Plants with Chitinase and Ribosome Inactivating Protein Genes and Their Resistance Identification

Soybean is one of the most important sources of edible oil and protein in the world. However, it suffers from many kinds of fungal diseases, such as frogeye leaf spot (Cercospora sojina) and soybean Phytophora root rot (Phytophthora megasperma Drechs). Fungal disease is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. Recently bean chitinase gene (chi), barley ribosome-inactivating protein gene (rip) and β-1,3-glucan have been successfully introduced to several plant species and achieved increasing resistance to several fungal diseases. In this study, plant expression vector containing bivalent fungi-resistance gene chi and rip was constructed and these two genes were transferred into four main soybean cultivars in Northeast China by Agrobacterium-mediated transformation. The identification of T₀ regenerated plants was determined by PCR and Southern blot analysis, transgenic soybean co-integrated the two anti-fungal protein genes were obtained, the transformation frequency was 0.5%. Four T₂ Bivalent transgenic soybean lines G0421, G0422, G0423 and G0424 were obtained and the resistance to P. megasperma and C. sojina of the four lines were characterized. Comparing to the non-transgenic type which was sensitive to P. megasperma, G0421, G0422, G0423 were resistant and G0424 was moderately resistant to P. megasperma. All the four lines were resistant to C. sojina while the non-transgenic type is moderately resistant to C. sojina. PCR amplification demonstrated that the T₂ bivalent transgenic soybean lines contained the two transferred genes. RT-PCR analysis showed that both of the two transferred genes expressed in the transgenic soybean lines. These transgenic soybean lines with increasing resistance to P. megasperma and C.sojina will be useful in soybean resistance breeding.