转Cry1Ab-t基因玉米YA108的获得及其抗虫性鉴定  

Acquirement and Bioassay of Transgenic Insect-resistant Cry1Ab-t Gene Maize YA108

作　　者：岳润清[1] 刘璐[1] 铁双贵[1] 徐心志[1] 陈娜娜[1] YUE Runqing;LIU Lu;TIE Shuanggui;XU Xinzhi;CHEN Nana(Cereal Crops Research Institute, Henan Academy of Agricultural Sciences,Zhengzhou 450002, China)

机构地区：[1]河南省农业科学院粮食作物研究所,河南郑州450002

出　　处：《华北农学报》2021年第2期212-218,共7页Acta Agriculturae Boreali-Sinica

基　　金：转基因生物新品种培育重大专项(SQ2019ZD080072);河南省农业科学院杰出青年科技基金(2020JQ01)。

摘　　要：为了培育优良的抗虫玉米自交系,通过农杆菌介导的方法,将含有自主改造合成的抗虫基因Cry1Ab-t的植物表达载体pCAMBIA3300m导入玉米杂交种HiII基因组中,通过双丙氨磷筛选、PCR检测获得阳性转基因植株,采用RT-PCR、试纸条、ELISA方法检测外源基因的表达情况,并进行抗虫性鉴定。结果表明,用双丙氨磷筛选后,获得了116株转基因玉米植株;经目的基因Cry1Ab-t和标记基因Bar的PCR检测,获得转基因阳性植株82株。选取长势好的转基因材料YA108进行RT-PCR、Bt-Cry1Ab/Ac试纸条和ELISA分析发现,转基因玉米YA108的Cry1Ab-t基因在转录、翻译水平上均表达。玉米花丝接虫试验表明,非转基因玉米花丝虫食严重,表现为高感玉米螟;而转基因玉米YA108花丝抗虫效果明显,均达到高抗水平,抗虫性表现稳定;吐丝期接虫试验表明,非转基因玉米接虫后期被咬食严重,茎秆多处有蛀孔,伤口处大多有霉菌,而转基因玉米YA108没有受到危害。田间接虫后,无论叶片和花丝,转Cry1Ab-t基因抗虫玉米材料YA108对亚洲玉米螟都具有非常好的抗性,能够短时间杀灭玉米螟幼虫,并极大程度减少了由于虫害引起的霉变。室内和田间接虫鉴定结果表明,转基因玉米YA108对亚洲玉米螟有较强的杀虫效果,田间抗虫等级为1级,抗性水平为高抗。In order to breeding maize inbred lines with favorite pest resistant.The modified insect resistant gene Cry1Ab-t was constructed into the plant expression vector pCAMBIA3300m and transferred into the hybrid maize HiII immature embryo with the Agrobacterium-mediated method.The positive transgenic plants were obtained by bar screening and PCR detection.RT-PCR,test strip and ELISA methods were used to detect Cry1Ab-t gene expression in transgenic maize,and the insect resistance was evaluated.The results showed that one hundred and sixteen seedlings were obtained after selecting with bialaphos sodium,and eighty-two seedlings of them were positive by PCR analysis and detection of the Cry1Ab-t gene and bar marker gene.RT-PCR,BT-Cry1Ab/Ac test strip and ELISA methods were used to detect Cry1Ab-t gene expression in transgenic material YA108 with good growth,Cry1Ab-t gene was expressed at transcription and translation level.The results of insect identification indicated that the non transgenic maize filaments were seriously infested by corn borer.However,the transgenic maize YA108 had a high level of insect resistance and stable insect resistance.The non transgenic maize was bitten seriously in the late stage of silking,and there were many boreholes in the stem and mold in the wound,while the transgenic maize YA108 was not damaged.After field inoculation,YA108,the Cry1Ab-t transgenic maize,had good resistance to Asian corn borer in both leaves and filaments.It could kill the larvae of Asian corn borer in a short time and reduce the mildew caused by insect pests to a great extent.The results of indoor and field indirect insect identification showed that the resistance rate of YA108 was detected to be Class 1 at each generation,showing high resistance level to Ostrinia furnacalis.

关 键 词：转基因玉米 Cry1Ab-t基因 农杆菌介导 分子鉴定 玉米螟 抗虫鉴定 

分 类 号：S513[农业科学—作物学] Q78[生物学—分子生物学]