转双价抗虫基因Bt-pta玉米植株的获得

以7922、8902、4112、340和853为受体材料,研究不同基因型的愈伤组织诱导与再生芽苗的关系,其中7922芽苗率为76.4%;8902、4112、340芽苗率分别为8.2%、12.7%和0%和11.8%,结果显示7922主要以体胚发生为主,芽苗率高,是遗传转化的良好受体材料。且胚性愈伤组织随着继代次数的增加变异率随之增加,应尽量减少继代次数以提高转化率。研究进一步利用PDS-100型基因枪将包含抗除草剂基因(bar)、苏云金杆菌毒蛋白基因(cryIA(a))和半夏凝集素基因(Pinelliaternataagglu-tinin,PTA)的聚合载体p3300-bt-pta导入7922的胚性愈伤组织,经PPT(3mg/L)筛选,获得抗性愈伤组织和再生植株。从获得的28株再生植株中筛选获得22株具有除草剂抗性的植株,PCR分析表明有7株同时含有Bt、PTA、Bar三个基因,共整合的频率为31.8%,结果表明将多价抗性基因同时导入玉米获得多抗的转基因玉米材料是可行的,这为通过转基因聚合育种培育创造同时抗鳞翅目害虫和同翅目害虫的玉米品种奠定了基础。

Synchronous Expression of pta and CryIA(a) Gene in Maize

Different genotype of maize inbred lines (7922,8902,4112,340,853) was used to study the relationship between somatic embryo formation and regeneration frequency.Results showed that 7922 had a regeneration frequency of 74.4% in somatic embryo pathway,while 8902,4112,340 and 853 had a regeneration frequency of 8.2%,12.7%,0%,11.88% in adventitious buds pathway.It concluded that 7922 was the best genetic transformation receptor in this experiment.Mutation frequency would be accumulated with increasing somatic callus subculture times;therefore,subculture times should be decreased for high transformation frequency.A aggregated construct with bar,cryIA(a) and pta,named p3300-bt-pta,was transformed into 7922 calli by PDS-100.Herbicide (phosphinothricin,PPT3mg/L) resistance screening indicated that there were 22 individual putative transformed plants with herbicide resistance in total.PCR detection further revealed that there were 7 individuals with three resistant genes integrated and cointegration frequency was 31.8%.In conclusion,it is feasible to introduce multi-resistant-genes into maize to get transgenic maize with multi-resistance to Lepidoptera insect and Homoptera insect in a single transgenic event.