转Cry1Ac青花菜回交后代鉴定方法研究  

Studies on Identification Methods in Backcross Generation of Cry1Ac-transgenic Broccoli

作　　者：李占省[1] 张黎黎[1] 张小丽[1] 舒金帅[1] 苏彦宾[1] 孙继峰[1] 刘玉梅[1] 方智远[1] 杨丽梅[1] 庄木[1] 张扬勇[1] 吕红豪[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所,农业部园艺作物生物学与种质创制重点实验室,北京100081

出　　处：《园艺学报》2018年第1期97-108,共12页Acta Horticulturae Sinica

基　　金：“十二五”国家科技支撑计划课题项目(2013BAD01B04); 国家现代农业产业技术体系建设专项资金项目(CARS-23-A); 中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS)

摘　　要：转Bt基因青花菜在遗传分离后代中的鉴定方法,对于准确筛选阳性单株和抗虫材料具有重要影响。利用5份青花菜材料、1份阳性对照(转Cry1Ac基因纯合材料)和5份回交父本(阴性对照)为试验对象,在基因水平上对Cry1Ac进行普通PCR扩增,蛋白水平上采用Bt-Cry1Ab/1Ac转基因检测试纸条测定,表观上采用小菜蛾离体饲虫试验进行抗性鉴定,比较阐明了3种方法的鉴定效果和优缺点。结果表明,3种方法分别从5份青花菜回交材料的159个分离单株中检测到82、77和75份阳性株,经卡方显著性检验,各材料阳性单株与非阳性单株分离后代均符合孟德尔遗传规律。分析表明,小菜蛾离体饲虫试验对于鉴定转Cry1Ac后代抗虫效果准确可靠,但需要很好地控制实验条件;Bt-Cry1Ab/1Ac转基因检测试纸条灵敏度高,可快速观察到Bt毒蛋白的实际表达量,基本不受条件限制,但必须控制人为误差;普通PCR扩增能够鉴定阳性株,但不能反映实际抗虫效果,必须结合小菜蛾饲虫试验或Bt毒蛋白水平上的检测才能实现精准鉴定。It’s important for separation offspring in transgenic Bt gene broccoli to identify positive plant lines and resistant materials accurately by different methods.In the study,five backcross materials based on four to five generations,one homozygous material as positive control and five inbred lines as negative controls were used for analysis of the insect-resistant effectiveness of Cry1Ac in transgenic broccoli from the levels of gene,protein and appearance.The gene of Cry1Ac was carried out by PCR amplification,protein was carried out by determination of Bt-Cry1Ab/1Ac test strip,and appearance was carried out by feeding test of diamondback moth in vitro.At the same time,the advantages of three methods were evaluated and stated.The result showed that a total of 82,77 and 75 positive strains were divided from 159 strains of five backcross materials.According to chi-square test at the level of 5%,the positive and none positive strains of 1︰1 were calculated and in accordance with mendelian inheritance.By comparisons of three methods,we found that the feeding trial of diamondback moth in vitro was accurate and reliable,but the experimental conditions must to be well controlled.The Bt-Cry1Ab/1Ac test strip was sensitive,fast and can observe the actual Bt protein expression,but it must control human errors.The normal PCR amplification could be used to identify the positive strains,but it can not reflect the actual anti-insect effect,so it needs to be together with the test of feeding diamondback moth or the test of Bt protein,which could provide an accurate result.

关 键 词：青花菜 转基因 CRY1AC 小菜蛾 遗传 

分 类 号：S635.9[农业科学—蔬菜学]