Bt抗虫水稻转育后代两种筛选方法的比较

Comparison of two screening methods for detecting insectresistant offspring of Bt-transgenic rice

【目的】通过比较分子标记辅助选择和除草剂抗性筛选两种检测方法的优缺点,为快速、准确、经济有效地选择抗螟虫水稻转育后代提供依据。【方法】用分子标记检测和除草剂抗性筛选两种方法,对不同组合的抗螟虫转育后代进行筛选,同时采用ELISA检测方法对筛选的抗性株系测定杀虫蛋白含量,比较两种筛选方法的准确性。【结果】在以T1C-19为供体亲本的4个组合169个单株中,两种筛选方法的符合率达到98.2%;而以T2A-1为供体亲本的3个组合141个单株中,两种筛选方法的符合率达到98.6%。在随机选择的经两种筛选方法鉴定均为阳性的144个单株中,均含有对应Bt抗虫基因的杀虫蛋白表达量,与亲本T1C-19、T2A-21相比,Bt抗虫水稻转育后代中个别单株的Bt蛋白含量出现大幅度下降或上升的现象。【结论】分子标记检测和除草剂抗性筛选对抗螟虫水稻转育后代的选择均有很高的准确性:分子标记检测准确可靠,且不受环境影响,但费用相对高;除草剂抗性筛选法耗费低、简便易行,但易受雨天影响。在筛选的过程中可根据现有的条件做出适宜选择。

[Objective]Two screening techniques, viz., marker-assisted selection and herbicide resistance screening were compared to develop an efficient and accurate selection method for detecting insect-resistant offsprings of Bt-transgenic rice. [Method]In the first method, two DNA markers linked to Cry1C^＊ and Cry2A^＊ genes were respectively used to screen the genomic DNA extracted from seedlings of the transgenic offspring plants. In the second method, adult plant leaves were sprayed with 300 mg/L herbicide Bastar and the withered and yellow leaves were counted to calculate the rate of positive plants harboring target genes. Moreover, the Bt protein content in the resistant plants was quantified using ELISA in order to verify the accuracy of two screening methods. [Results]The consistency rate of two screening methods was 98.2% in screening of 169 individual plants generated from 4 combinations by using T1C-19 as donor parent. In screening of 141 individual plants generated from 3 combinations by using T2A-1 as donor parent, the consistency rate of two methods was 98.6%. The ELISA assay results showed that the Bt protein in 144 individually selected plants （from positive plants）, using both of the two methods, had corresponding expression and was increased or decreased substantially compared to donor parents T1C-19 and T2A-21. [Conclusion]Both the marker-assisted selection and herbicide resistance screening techniques were highly accurate in selecting insect-resistance offspring of Bt-transgenic rice. The former one can be used in lab at any stage but the cost was relatively higher, while the latter one is cost-effective and convenient, however, easy to be affected by environmental factors. In general, this study provides useful information for breeding Bt-transgenic rice.