转基因复合抗虫耐除草剂玉米LD05的分子特征及抗性评价  

Molecular Characteristics and Resistance Evaluation of Transgenic Maize LD05 with Stacked Insect and Herbicide Resistance Traits

作　　者：岳润清 李文兰[1] 丁照华[1] 孟昭东[1] YUE RunQing;LI WenLan;DING ZhaoHua;MENG ZhaoDong(Maize Research Institute,Shandong Academy of Agricultural Sciences/Shandong Key Laboratory of Maize Biological Breeding/National Engineering Center of Wheat and Maize,Jinan 250100)

机构地区：[1]山东省农业科学院玉米研究所/山东省玉米生物育种重点实验室/小麦玉米国家工程中心,济南250100

出　　处：《中国农业科学》2025年第7期1269-1283,共15页Scientia Agricultura Sinica

基　　金：泰山学者工程;泰山产业领军人才;山东省玉米产业技术体系(SDAIT-02-04);山东省良种工程(2023LZGC003);国家现代农业产业技术体系(CARS-02-09);山东省农业科学院创新工程(CXGC2024F02)。

摘　　要：【目的】明确转基因复合抗虫耐除草剂玉米LD05的分子特征和目标性状有效性,为产业化应用提供数据基础、技术支撑和产品储备。【方法】利用生物信息学分析设计改造获得的自主产权抗虫融合基因m2cry Ab-vip3A,针对创制的新型转基因复合抗虫耐除草剂玉米LD05,选取其BC_4F_3、BC_4F_4、BC_4F_5代开展试验研究。利用特异性PCR和Southern blot开展基因组整合稳定性分析;利用q RT-PCR和ELISA开展表达稳定性分析;通过室内生测和田间接虫试验评价其对靶标害虫的抗性,通过田间喷施草铵膦测试其除草剂的耐受稳定性。【结果】发掘设计出自主产权新型抗虫融合基因m2cry Ab-vip3A,创制出复合抗虫耐除草剂玉米转化体LD05,其外源T-DNA以单拷贝形式整合进玉米基因组中。q RT-PCR结果表明,m2cry Ab-vip3A和bar在3个世代不同组织器官中均有表达,表达量变化趋势基本一致。其中,m2cry Ab-vip3A在连续3个世代苗期叶中表达量最高,平均表达量为36.73,而在成熟期穗轴中表达量最低,平均表达量仅有0.91;bar与m2cry Ab-vip3A表达模式相似,在苗期叶片中表达量最高,平均表达量为7.35,在拔节期以后表达量变低。ELISA结果表明,M2Cry Ab-VIP3A在3个世代不同时期、不同器官中均能稳定积累,且不同世代蛋白积累量相似,其中,不同世代苗期叶中积累量最高,均高于19.67μg·g^(-1) FW;PAT蛋白在连续3个世代的不同组织中均能检测出较高目标蛋白的表达,且在不同世代之间表达量无明显差异,其中,在不同世代苗期叶中表达量最高,平均含量为16.61μg·g^(-1) FW,而在成熟期根中的积累量最低,平均含量为0.30μg·g^(-1) FW。室内生测结果显示,取食LD05的心叶期玉米叶片组织96 h后,玉米螟、草地贪夜蛾、黏虫的校正死亡率均达到100%,为高抗级别;花丝期接虫96 h后,玉米螟、草地贪夜蛾、棉铃虫的校正死亡率分别为100%、100%和98.67%,均为�【Objective】To clarify the molecular characteristics and the effectiveness of target traits of transgenic maize LD05 with composite insect and herbicide resistance,and to provide data basis,technical support and product reserve for industrial application.【Method】Using biological information analysis,we designed and modified the proprietary insect-resistant fusion gene m2cryAb-vip3A,and selected BC4F3,BC4F4 and BC4F5 generations of the newly created transgenic hybrid insect-resistant and herbicide-tolerant maize LD05 to carry out experimental research.Specific PCR and Southern blot were used to analyze the stability of genomic integration.qRT-PCR and ELISA were used to analyze the expression stability.The resistance to target pests was evaluated by bioassay and field trials,and the herbicide tolerance was tested by field spraying of glufosinate.【Result】A new insect-resistant fusion gene m2cryAb-vip3A with independent property right was discovered and designed,and a multivalent insect-resistant and herbicide resistant maize transformant LD05 was created.The exogenous T-DNA was integrated into the maize genome in the form of a single copy.The qRT-PCR results indicated that m2cryAb-vip3A and bar were both expressed in various tissues and organs across three generations,and the variation trend of expression quantities was largely consistent.Specifically,the expression level of m2cryAb-vip3A was the highest in the leaves at the seedling stage of the three consecutive generations,with an average expression quantity of 36.73,while the expression level was the lowest in the cob at the mature stage,with an average of merely 0.91.The expression pattern of bar was similar to that of m2cryAb-vip3A,with the highest expression level in the leaves at the seedling stage,averaging 7.35,and the expression level decreased after the jointing stage.The ELISA results demonstrated that M2CryAb-VIP3A could stably accumulate in different organs and at different periods in the three generations,and the protein accumulation amoun

关 键 词：转基因玉米 复合性状 m2cryAb-vip3A BAR 分子特征 抗性评价 

分 类 号：S435.1[农业科学—农业昆虫与害虫防治]