抗虫基因cry1Ab13在大豆中的遗传转化及抗虫性鉴定

Identification on Insect-resistant Gene of cry1Ab13 Transformation into Glycine max L.Merr.

cry1Ab13基因是抗虫基因,编码的杀虫晶体蛋白对鳞翅目害虫具有一定的毒杀作用,构建了植物表达载体pCambia3300-35S-cry1Ab13,通过花粉管通道法将该载体转入大豆品种吉农28中,经PCR扩增检测得到15株T_1代阳性植株。Southern blotting分析显示有5株出现杂交信号,并以单拷贝形式整合到大豆基因组中。荧光定量PCR测定结果表明:cry1Ab13基因在转化植株的叶、茎秆中均有表达,每株表达量各不相同,在叶片部位表达量相对较高,最高为6.7,最低为2.5;在茎部表达量最高为0.76,最低为0.31。对T_1代阳性植株籽粒,采用圆盘分隔法接入大豆食心虫幼虫,进行初步抗虫试验,结果显示转化植株抗虫效果明显,但后代稳定性还需进一步研究。

The crylAbl3 gene used in this experiment is the insect resistant gene, it encodes insecticidal crystal protein and has certain toxic killing effect on Lepidoptera pests. The plant expression vector of pCambia3300-35S-crylAb13 was constructed. The transgenic plants were obtained by pollen-tube pathway transformation of soybean JN28. 15 transformed plants in T1 generation were tested by PCR, while Southern blotting indicated that only 5 of them showed hybridization bands, and the functional fragment was integrated into the soybean genome by single copy. The result of quantitative real-time PCR showed that the crylAbl3 gene was expressed in soybean leaves and stems, and each plant expression was different ,the relatively high expression was in leaves,the highest was 6. 7, the lowest was 2. 5, while it was 0. 76 and 0. 31 in the stems. The seeds of PCR positive plants in T1 generation and newly hatched larvaes were introduced, and initial insect resistant assay was made by disk method. Result suggested that transgenic plant had an obvious resistant effect. Further investigations on the genetic stability of crylAbl3 in transgenic plants and generations are to be carried out.