农杆菌介导甜高粱转Bt cry1Ah的研究

Introduction of Bt cry1Ah Gene into Sweet Sorghum (Sorghum bicolor L.Moench) by Agrobacterium tumefaciens-Mediated Transformation

【目的】建立一套高效、稳定的甜高粱农杆菌遗传转化体系,并在甜高粱中验证来源于苏云金芽胞杆菌新型杀虫基因cry1Ah表达产物的杀虫活性。【方法】以甜高粱幼穗愈伤组织为转化受体,通过农杆菌介导法将密码子优化过的Bt cry1Ah导入2个甜高粱品种"BABUSH"和"MN-3025"中。对获得的再生植株进行PCR检测、RT-PCR分析及除草剂抗性、目的蛋白表达水平和抗虫性鉴定分析。【结果】对336块幼穗愈伤组织块进行了农杆菌侵染,经双丙氨膦(Bialaphos)梯度筛选后共获得66株再生植株,PCR鉴定在8个转化事件中有22株为阳性植株,平均转化率为2.38%。RT-PCR检测结果表明,cry1Ah在T0甜高粱转基因植株中能够正常转录。Western blotting分析和ELISA定量测定显示,有5株可检测到Bt蛋白,但Bt蛋白含量差异较大,最高可达165.69 ng.g-1叶片鲜重(FW),最低的仅为1.93 ng.g-1叶片鲜重(FW),平均为87.50 ng.g-1 FW。饲虫试验表明,有2株转基因甜高粱植株对亚洲玉米螟(Ostrinia furnacalis)表现为抗性。【结论】利用建立的以甜高粱幼穗为外植体的农杆菌遗传转化体系,可获得具有玉米螟抗性的转基因甜高粱植株,其后代遗传稳定性还有待于进一步的研究。

【Objective】 The aim of the study is to establish a high frequency and steady Agrobacterium tumefaciens-mediated transformation system of sweet sorghum,and validate the insecticidal function of a novel Bt cry1Ah gene in the transgenic plants.【Method】Using the callus induced from immature inflorescence as transformation recipients,the codon optimized Bt cry1Ah was transferred into sweet sorghum varieties ＂BABUSH＂ and ＂MN-3025＂ via Agrobacterium-mediated transformation.The obtained regenerative plants were identified by PCR,RT-PCR analysis,and their herbicide resistance,the expression of aim protein and insect-resistant identification were also analyzed.【Result】After gradient selection with Biolaphos,a total of 66 regenerated plants were produced from 336 agroinfected calli in these two sweet sorghum varieties.Among these plants,22 PCR positive transformed plants of 8 independent transformation events were obtained,and the average transformation efficiency was 2.38%.The transcription of cry1Ah gene in the T0 transgenic sweet sorghum plants was further confirmed by RT-PCR.The Bt proteins could be detected by western blotting and ELISA assay in five transgenic plants,which showed different expression levels in a range of 1.93 ng？g-1 FW to 165.69 ng？g-1 FW,with an average of 87.50 ng？g-1 FW.Additionally,the results of bioassay indicated that two of the five transgenic plants displayed high insect-resistance to Ostrinia furnacalis.【Conclusion】An Agrobacterium-mediated genetic transformation system of sweet sorghum was established by using the callus derived from immature inflorescence as the recipients.The resulted T0 generation transgenic sweet sorghum plants with cry1Ah showed high insect-resistance to Ostrinia furnacalis.Further investigations on the genetic stability of cry1Ah in different transgenic lines and generations are undergoing.