摘要
菌核病是我国油菜主产区的主要病害 ,系由核盘菌 (Sclerotiniasclerotiorum)的子囊孢子在晚春直接侵染开花植株衰老的叶片 ,或先侵染飘落在衰老叶片上的花瓣后再产生菌丝侵入叶片和植株本体。几丁质是核盘菌菌丝体细胞壁的主要组成成分 ,几丁质酶破坏真菌菌丝尖端新合成的几丁质而抑制病菌的生长。草酸是核盘菌侵染寄主植物后分泌的毒素 ,草酸氧化酶能够分解草酸 ,缓解核盘菌在油菜体内的毒性。我们将几丁质酶基因和草酸氧化酶基因与拟南芥衰老叶片启动子PSAG12 嵌合在一起构建成表达载体 ,使两外源抗病基因仅在转基因油菜的衰老叶片中表达 ,在发挥抗病作用的同时最大限度地节约了植物的能量消耗。本实验采用inplanta的转化方法 ,转化效率在 0 1%左右 ,每个转基因植株的插入位点为 1- 3个拷贝。对T1代转基因植株的RT PCR分析表明 ,两个外源抗病基因在衰老叶片中的表达强于幼嫩叶片。转基因植株叶片尤其是衰老叶片对核盘菌的抗性及对草酸毒素的耐性均得到了增强。从转基因植株上飘落的花瓣也表现出对核盘菌抗性的提高。抗病双基因在叶片和花瓣发育的特定时期表达不仅提高了油菜对核盘菌侵染的针对性 ,还可能减轻转基因植株的能源消耗和生理扰乱。对转基因植株后代的农艺学和生理学研究正在进行之中。
Fungal pathogen Sclerotinia sclerotiorum invades senescence leaves of flowering plants of B.napus directly or mediated by the petals dropped on the leaves in later spring in the main area of rapeseed production in China. While chitinase degrades fungal cell wall structural polysaccharides to inhibit fungal growth, oxalate oxidase degrades oxalic acid which is harmful to rapeseed plants. Dual genes coding for these two enzymes, ChiB and the OxO, were constructed under a leaf senescence associate promoter, SAG12, respectively and transformed into B.napus with in planta method. Hundreds of transformed seedlings were obtained with a frequency of 0.1% and each transformants has 1 to 3 insertion sites. RT-PCR performance with T 1 plants showed that the transgenes express strongly in senescence leaves. Both Sclerotinia resistance and oxalic acid tolerance were significantly increased in senescence leaves of T 1 lines. Newly emarcid petals also shown increased ability to Sclerotinia invasion into plants. Dual gene expression in leaves and petals at right time enhanced the distinction of resistance to Sclerotinia in rapeseed. Further investigations and improvements on T 2 homozygous lines are discussed.
出处
《分子植物育种》
CAS
CSCD
2003年第3期303-312,共10页
Molecular Plant Breeding
