Ri质粒介导的几丁质酶基因转化小麦的研究

采用L₉（3⁴）的正交试验，以带有pMLH7133-Chi质粒的发根农杆菌为介导，对栽培品种春小麦东农7742、龙麦9814和Hite的幼胚愈伤组织进行了遗传转化研究。结果表明：基因型为东农7742、菌液浓度OD₆₀₀=0.8、共培养时间2 d、乙酰丁香酮（As）浓度为100 μm是最佳的试验组合；各影响因素的主次顺序是：基因型＞乙酰丁香酮的浓度＞菌液浓度＞共培养时间。共获得138株Hyg抗性植株，经PCR及PCR-Southern检测初步证明，外源几丁质酶基因已整合到其中5株的基因组中，又对3株阳性植株进行了RT-PCR扩增和几丁质酶活力测定，证明外源几丁质酶基因已在小麦基因组中稳定表达。

Chitinase gene mediated by Ri plasmid transfering wheat

Mediated by Agrobacterium rhizogene which contains plasmid pMLH7133-Chi, the genetic transformation experiment was carried out with the callus of Spring Wheat Dongnong 7742, Longmai 9814, and Hite as the acceptor in the way of orthogonal test of L₉(3⁴). The result indicates that the best group is Dongnong 7742, 100 μm of Acetosyringone, A. rhizogene’s OD₆₀₀=0.8 and about two days’ co-cultivation; the order of influential factors is: genotype>concentration of Acetosyringone>concentration of A. rhizogene> co-culture period. 138 regenerated plants were obtained in the experiment. Then detected by PCR amplification and PCR-Southern, the result suggests that the foreign Chitinase genes have already been introduced into 5 wheat genomes of all. Three of the five plants were detected again by RT-PCR and Chitinase activity methods, which proved that the foreign Chitinase gene has expressed steadily in wheat genome.