转几丁质酶和β-1,3-葡聚糖酶双价基因小麦的获得和鉴定

Development of Transgenic Wheat Plants with Chitin ase and β-1,3-Glucosanase Genes and Their Resistance to Fusarium Head Blight

利用农杆菌介导法将几丁质酶基因和β1,3 葡聚糖酶双价基因导入小麦品种扬麦10号和Bobwhite ,共得到了13株T0 代转基因植株,2个小麦品种的转化效率分别为1 .4 %和1. 0 %。T1 代植株赤霉病抗性鉴定结果表明,转基因植株对赤霉病具有一定抗性,小穗发病率4 .76 %～11. 76 % ,低于抗病对照和感病对照。T1 代植株分子检测结果表明,外源基因分离比例为2. 33∶1,较低的分离比例可能与外源基因的丢失有关。

Most wheat diseases are caused by fungi pat hogens, such as scab, powdery mildew, rust and take all. Twenty to thirty ye a rs ago, scab mainly happened in Southeast China, but it has extended into nort hwest and northeast China in the latest two decades. In normal years, this dis ease makes wheat production lost by 10 percent to 20 percent, but 30 percent in its popular years. However, it is very difficult to develop resistant materia ls to such kind of disease only by traditional breeding technique because of the limited resistant resource in wheat, even though local variety Sumai 3 has bee n efficiently used in commercial breeding for many years. Therefore, it is ver y important to transfer some genes related to the resistance of scab into wheat by genetic transformation. Chitinase and β-1,3-glucosanase genes were prove d to be useful in controlling fungi diseases at different extent in various plan ts. The purpose of this research is to introduce chitinase and β-1,3-glucos anase genes into wheat for improving its resistance to scab. Based on such a ta rget, chitinase gene and β-1,3-glucosanase gene side by side (called as G CE) were constructed into binary vector pCAMBIA3301 (Fig.1), and then transf ormed into C58c1 Agrobacterium strain. The immature embryos isolated from Y angmai 10 and Bobwhite were pre-cultured for four days on callus induction medi um, and then infected by the Agrobacterium strain. After co-cultivation s teps, the explants were cultured on selection medium with 10-25 mg/L G418 for callus induction and plant regeneration in order. Identified by leaf painting a nd PCR analysis, 13 T 0 transgenic plants were finally obtained from the two v arieties, with transformation frequencies of 1.4% and 1.0%, respectively. T 1 plants were further tested for genetic analysis and scab resistance. Southe rn blot showed that the alien genes were integrated into wheat chromosome by 1- 2 copies in most transgenic plants (Fig.5). Northern blot showed that the alie n genes were transcribed stably into RNA almost in all transgenic plants (Fig.6 ). PCR result indicated that the segregation ratio of the alien genes was 2.33 ∶1 in the T 1 progeny (Table 1), and the low rate might be related to the los ing of the foreign genes. Field test showed that five transgenic lines of GCE- 6, GCE-11, GCE-13, GCE-16, and GCE-21 appeared good resistance to Fusari um head blight, and the infected floret frequency with the pathogen varied from 4.76% to 11.76% (Table 2). The GCE gene performed scab resistance in whe at according to this study, and the transgenic materials obtained can be potent ially used in wheat scab resistance breeding.