【作者】 岳同卿；

【导师】 黄大昉；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 Bt cry1Ah基因是本实验室从国内苏云金芽胞杆菌(Bacillus thuringiensis, Bt)分离株BT8中克隆的新型杀虫蛋白基因,其编码蛋白对鳞翅目害虫具有强毒力,尤其对亚洲玉米螟(Ostrinia furnacalis )的毒力强于目前转基因作物研究中使用的cry1A类基因。利用cry1Ah基因进行抗虫转基因玉米研发在提高转基因玉米抗虫性的同时可以降低害虫抗性风险,获得的转基因玉米具有很好的应用前景。为了在玉米内验证cry1Ah的功能,本研究首先利用cry1Ah基因构建了植物表达载体pHUAh,该载体以潮霉素磷酸转移酶基因(hygromycin phosphotransferase,hyg)为筛选标记基因。用基因枪法转化玉米Q31(齐31)和Z31(综31)杂交组合的胚性愈伤组织,得到66株T0代转化植株,分子检测确定13株为阳性转基因玉米植株。通过对T1代植株进行田间抗虫性检测,筛选得到两个抗性转基因事件B1和B7。对B1和B7的T1-T5代转基因植株进行了四年跟踪检测,结果表明cry1Ah基因在玉米植株中可以稳定表达,并且可以逐代稳定遗传。转化cry1Ah基因的玉米对玉米螟具有较好的抗性,该基因可继续用于转基因抗虫玉米的研发。随后,我们利用cry1Ah基因构建了一个无筛选标记基因的植物重组表达载体pUAh,利用花粉管通道法转化玉米自交系X090。对获得的2200株T1代转化植株的Bt Cry1Ah蛋白表达进行检测,有172株为阳性植株。通过田间抗虫性测定,筛选到35株抗性植株。对其T2代植株进行抗虫性测定后确定了12个高抗转基因事件。随后分别从DNA水平、蛋白水平及田间抗虫性三方面对这12个高抗转基因事件的T2-T5代转基因植株进行了跟踪检测:DNA水平分析发现cry1Ah在转基因玉米中具有很好的遗传稳定性,T5代有9个转基因事件已经纯合,PCR阳性率为100%。蛋白水平的分析结果表明该基因在每代都可以正常稳定表达,但Cry1Ah蛋白在不同转化事件之间及同一转化事件不同组织部位内的表达量存在差异:转化事件P12(P12-8-7-13行)的Cry1Ah平均表达量最高,占可溶性总蛋白的0.130%。转化事件P16(P16-16-6-5行)的Cry1Ah平均表达量最低,占可溶性总蛋白的0.023%。对P12不同组织部位的Bt蛋白表达量的检测结果表明,Cry1Ah在苞叶中的表达量最高,占可溶性总蛋白的0.186%。抗虫性研究结果表明转cry1Ah基因玉米对亚洲玉米螟具有显著抗性,抗性可以逐代稳定遗传,并且抗性与蛋白的表达水平呈正相关。此外,本研究利用TAIL-PCR技术对花粉管通道法获得的转cry1Ah抗虫玉米P19的外源基因整合位点3′侧翼序列进行了初步研究,对得到的3′侧翼序列进行序列比对后发现其与玉米基因组4号染色体上的一段序列同源性最高,核苷酸一致性为99.5%。在玉米基因组内的具体位置为BIN4.09,初步确定了外源基因在玉米基因组中的整合位点。本研究获得的无筛选标记的遗传稳定的转基因玉米有很好的应用价值,可以作为候选材料进行下一步Bt抗虫玉米的育种工作。更多还原

【Abstract】 The cry1Ah gene was one of the novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8 which exhibited high toxicity against lepidopteran larvae. Cry1Ah protein showed higher toxicity to Asian corn borer (Ostrinia furnacalis) than any other cry1A genes. Transferring cry1Ah gene into maize can not only improve the resistance of transgenic maize to Asian corn borer but also delay the insect resistance risk. The cry1Ah gene was a candidate gene for insect resistant transgenic maize research.To confirm the cry1Ah gene’s function in transgenic maize, a plant expression vector pHUAh harboring the cry1Ah gene was constructed and transferred into immature embryonic calli of maize Q31×Z31 by microprojectile bombardment. The hyg gene was used as the selective marker gene. Sixty-six regenerated plants were obtained in T0 generation,thirteen of which were PCR positive. Bioassay results of T1 transgenic plants showed that events B1 and B7 were high resistant to the Asian corn borer . The two events were monitored by molecular detection and bioassay for 4 years from T1 toT5 generations. The results showed that foreign gene cry1Ah was expressed stably in maize and could be inherited stably over generations. Results of bioassay to T2-T5 transgenic maize plants suggested that the transgenic plants had high resistance against the Asian corn borer .A marker-free plant expression vector pUAh harboring the cry1Ah gene was constructed and transferred into maize inbred line X090 by pollen-tube pathway. Two thousands and two hundreds regenerated plants were obtained in T1 generation and were detected for the expression of Bt toxic Cry1Ah protein by Jin-biao immune detection strips. There were 172 plants expressing the Cry1Ah protein,35 of which were insect resistant confirmed through bioassay. In T2 generation, there were 12 transgenic events showing high resistance against the Asian corn borer which were further analyzed by DNA assay, protein assay and bioassay from T2 to T5 generations. Results of DNA analysis from T2 to T5 generations showed that foreign gene cry1Ah could be inherited stably over generations in transgenic maize. Nine homozygous transgenic events were obtained in T5 generation which showed uniformly positive in PCR assay. Protein analysis suggested that the Cry1Ah protein were stably expressed in each generation in transgenic maize and the contents of Cry1Ah protein were different among the 12 transgenic events as well as among different tissues. Content of Cry1Ah protein was the highest in transgenic event P12 ( line of P12-8-7-13 ) which was 0.130% of the total soluble protein . The lowest was the event P16(line of P16-16-6-5),of which the content of Cry1Ah protein was 0.023%. Detection of Cry1Ah protein in different tissues of event P12 showed that the Cry1Ah content was the highest in the husk leaf which was 0.186% of the total soluble protein. Bioassay results exhibited that the transgenic maize harboring cry1Ah gene showed high resistance against the Asian corn borer and the insect resistance could be inherited stably which were positively correlated with the level of the Cry1Ah expression. In this study, the integration 3’flanking sequence of cry1Ah gene in event P19 was analyzed by TAIL-PCR. The results showed that the flanking sequence was the highest homologous with the sequence at the BIN4.09 locus in the maize genome. The identity of nucleotide sequence was 99.5% .These revealed that foreign gene had been integrated into the maize genome.The stably inherited transgenic maize obtained in this study which had no selective marker gene could be potential candidates for the breeding of Bt insect-resistant transgenic maize.更多还原