将包含2个抗虫基因sbk\[修饰后的Cry1A(c)\]和sck（修饰后的CpTI）的质粒载体pCDMARUBAHyg转入农杆菌EHA105中, 感染南粳45的愈伤组织, 得到再生植株。用sbk和sck基因的引物进行PCR分析, 从97个再生植株中筛选出42个含有2个抗虫基因而没有潮霉素基因的阳性植株。通过Southern杂交发现, 42个阳性植株中具有1~5个外源基因拷贝的分别有23、 11、 5、2和1个单株。用RTPCR检测发现, 4个单拷贝株衍生的28个T3代植株外源基因能正常表达。人工饲喂二化螟幼虫试验也表明, 转基因植株的幼虫死亡率达94%~100%, 抗虫能力比对照显著提高。筛选出3个农艺性状优良的株系。
The plasmid of pCDMARUBAHyg, which contained two insectresistant genes, sbk\[modified from Cry1A(c)\] and sck(modified from CpTI), was transformed into an Agrobacterium EHA105 for infection of the calli of a super japonica rice Nanjing 45. Plants regenerated from the transformation were subjected to all kinds of gene verifications and selection. Using PCR detection with the primers of sbk and sck genes, 42 positive transgenic plants that were markerfree and contained the two target genes were selected from 97 regenerated plants. Results of Southernblotting indicated that 23, 11, 5, 2 and 1 plants had one, two, three, four and five copies of the transformed genes, respectively. Analyses of RTPCR and Bt gene testing paper showed that 28 T3 generation plants derived from four transgenic plants having a single copy were insectresistant. Feeding experiment with rice stem borer revealed that the insect resistance was greatly increased with nymph mortality ranging from 94% to 100%. In addition, among the transgenic plants, three T3 transgenic plants had many desirable characteristics for production. The mechanism of insectresistance of Bt gene and its application in rice transgenic research were also briefly discussed.
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