摘要
为获得新型抗虫转基因玉米,将通过群体筛选获得的具有抗虫性的转cry2Ah-vp基因玉米VP1-5采用PCR、Southern blot、实时荧光定量PCR(qPCR)、酶联免疫吸附测定(ELISA)等方法进行阳性植株鉴定、拷贝数分析、转录水平和翻译水平分析,同时通过室内和田间生物活性测定鉴定转基因玉米VP1-5对东方黏虫Mythimna separata和亚洲玉米螟Ostrinia furnacalis的抗性。结果表明,在转基因玉米VP1-5中cry2Ah-vp基因已整合到玉米基因组,以单拷贝的形式插入;cry2Ah-vp基因在转基因玉米VP1-5不同部位组织中均可以正常转录,在灌浆期叶片中的mRNA表达量最高,相对表达量为32.67,在灌浆期穗轴中的mRNA表达量最低,相对表达量为3.74;Cry2Ah-vp蛋白在转基因玉米VP1-5的6叶期各组织中表达量均较高,其中在叶片中的表达量达到2 155.18 ng/g FW,在抽雄期花丝中的表达量最高,达到2 165.86 ng/g FW;且转基因玉米VP1-5对东方黏虫有很高的杀虫活性,接虫3 d后幼虫死亡率达到100.00%;对亚洲玉米螟幼虫也有明显的生长抑制作用。表明转基因玉米VP1-5可作为玉米抗虫育种和害虫防治的种质资源。
To obtain the novel insect-resistant maize,the insect-resistant transgenic event VP 1-5 harboring cry2 Ah-vp gene was detected for the foreign gene insertion,copy number,transcriptional and translational expression of cry2 Ah-vp gene by using PCR,Southern blot,real-time fluorescence quantitative PCR(qPCR) and enzyme-linked immunosorbent assay(ELISA),and the bioassay in the laboratory and field was conducted to identify the resistance of VP 1-5 transgenic maize to Mythimna separata and Ostrinia furnacalis.The results showed that cry2 Ah-vp gene was integrated into maize genome with a single copy in transgenic maize VP 1-5.The cry2 Ah-vp gene was correctly transcribed in different tissues showing the highest expression in leaf and the lowest expression in cob during grain-filling stage,and the relative mRNA level were 32.67 and 3.74,respectively.The Cry2 Ah-vp protein was expressed relatively high in all tissues at six-leaf stage,especially in leaf where the expression was 2 155.18 ng/g FW.Overall,the highest expression of Cry2 Ah-vp protein was 2 165.86 ng/g FW in silk at tassel stage.VP 1-5 transgenic maize was demonstrated to have high insecticidal toxicity towards armyworm by the fact that the mortality of armyworm larvae reached 100.00% 3 d after inoculation;VP 1-5 obviously inhibited the weight gain of the corn borer larvae.This study indicated that VP 1-5 transgenic maize could be a good germplasm resource for insect resistance and pest control.
作者
李梦桃
李圣彦
汪海
张杰
苍晶
郎志宏
LI Mengtao;LI Shengyan;WANG Hai;ZHANG Jie;CANG Jing;LANG Zhihong(College of Life Sciences,Northeast Agricultural University,Harbin 150030,Heilongjiang Province,China;Biotechnology Research Institute,Chin ese Academy of Agricultural Sciences,Beijing 100081,China;State Key Laboratory for Biology of Plant Diseases and Insects Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China)
出处
《植物保护学报》
CAS
CSCD
北大核心
2020年第1期74-83,共10页
Journal of Plant Protection
基金
国家转基因生物新品种培育重大专项(2016ZX08003-001)
