AP70抗病基因转化番茄的研究

[目的]通过基因工程提高番茄抗病性。[方法]通过冻融法将含萝卜抗真菌蛋白基因的质粒AP70转入农杆菌LBA4404中,再用农杆菌介导法将AP70抗病基因对番茄进行遗传转化,研究预培养、侵染时间等因素对AP70转化番茄的影响。[结果]带柄子叶诱导番茄产生不定芽的效率比子叶块效率高。农杆菌浸染番茄子叶前预培养1 d,不仅能提高子叶存活率,而且可降低共培养时外植体的污染率。农杆菌对番茄子叶的侵染时间最好不超过5 min。外植体筛选培养基中Kam的浓度不宜太高或太低,可获得少量转AP70基因的抗性芽。[结论]农杆菌对番茄子叶侵染前要进行稀释,且侵染时间不宜超过5 min,共培养1 d时转化率较高。

Study on the Transformation of the Disease Resistance Gene AP70 into Tomato

[Objective] The aim of the research was to enhance the disease resistance of tomato through gene engineering.[Method] Plasmid AP70 containing Raphnus sativus-antifungal protein gene was transformed into A.tumefaciens LBA4404 by freeze-thawing method.And the disease resistance gene AP70 was used to make the genetic transformation of tomato by A.tumefaciens-mediated method to study the effects of such factors as preculture and the infection time on the tomato transformation with AP70.[Result] The efficiency of inducing tomato to produce the adventitious buds of the cotyledon with petiole was higher than that of cotyledon pieces.Preculture for 1 d before infecting tomato cotyledon by A.tumefaciens could not only increase the survival rate of cotyledon,but also reduce the pollution rate of explant in the stage of coculture.The infection time of A.tumefaciens on tomato cotyledon had better be not more than 5 min.Too high or low concn.of Kam in the screening medium of explant was not suitable and a few of the resistant buds transformed with AP70 gene could be obtained.[Conclusion] A.tumefaciens should be diluted before infecting tomato cotyledon.And its infection time should not more than 5 min and the conversion rate was higher after coculture for 1 d.