转双价抗虫基因大白菜新种质的获得及其抗虫性分析

Acquirement of Transgenic Chinese Cabbage with BmkIT-Chitinase Gene and Their Insect-resistant Bioassay

采用超声波辅助花粉介导方法,将双价抗虫基因BmkIT-Chitinase导入早熟型大白菜自交系20-19-3,最终获得了5个转基因大白菜优良自交系纯合株系Z1-5、Z2-7、Z9-6、Z11-6和Z20-13;以转基因大白菜株系和非转基因对照植株为材料,对BmkIT-Chitinase基因在大白菜中的遗传规律、基因表达及抗虫性进行进一步分析。结果显示:(1)转化株后代多代(T_1~T_4)PCR、Southern blotting等分子跟踪检测表明,目的基因已成功导入受体植株,且能够稳定遗传;用该转基因方法对大白菜进行基因转化所获得的转基因植株分析显示,外源基因多数以多拷贝形式整合于核基因组,少部分外源基因以单拷贝形式整合。(2)Elisa分析结果证明,所导入的外源基因可高效表达,T4代株系新鲜叶片中表达产物量最高达到0.069μg·g^(-1)左右。(3)转基因株系田间抗虫性统计分析表明,转化株系与对照在抗虫性方面有显著差异,其对小菜蛾及菜青虫抗性普遍提高2~3级。研究认为,转BmkIT-Chitinase基因大白菜中BmkIT-Chitinase基因的表达可有效提高大白菜的抗虫性。

At the present study,the binary insect-resistant BmkIT-Chitinase gene was successfully transformed into the Chinese cabbage inbred line 20-19-3via pollen mediated transformation approach assisted by supersonic.Five transgenic Chinese cabbage inbred lines Z1-5,Z2-7,Z9-6,Z11-6and Z20-13 were obtained.The genetic rules,transgenic expression and insect resistance of above transgenic materials were compared with untransformed control.The PCR amplification and Southern blot hybridization detection on transgenic plants and their progeny showed that：（1）BmkIT-Chitinase gene has been transformed into the genome of receptors and the transgenics can inherit stably.Most transgenics exist in multi-copy form and single copy transgenics appeared in few translines.（2）ELISA assay verified the expression of foreign gene and the expression product of BmkIT-Chitinase gene in T4 transgenic plants were high to 0.069μg·g^-1fresh leaves.（3）Field insect-resistant bioassay showed that there are significant differences on insect resistance between transformed inbred lines and their control groups.The level of insect resistance activities of Pieris rapae Linnaenus and Plutella xylostella Linnaenus enhanced 2-3degree.