Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi-284 003, India.
J Appl Genet. 2010;51(3):243-57. doi: 10.1007/BF03208854.
We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.
我们报告了一种无标记（“清洁 DNA”）转基因水稻（Oryza sativa）的产生，该水稻携带最小的基因表达盒，其中包含感兴趣的基因，并评估了其对黄条跳甲（Scirpophaga incertulas）（鳞翅目：Pyralidae）的抗性。这种转基因籼稻携带有两个不同的苏云金芽孢杆菌（Bt）基因的翻译融合体，即 cry1B-1Aa，由绿色组织特异性磷酸烯醇丙酮酸羧激酶（PEPC）启动子驱动。一个优秀的籼稻品种 Pusa Basmati-1 的成熟种子衍生愈伤组织与 Bt 基因和标记基因 hpt 的基因表达盒（清洁 DNA 片段）一起共轰击，以生成无标记的转基因水稻植株。用于轰击的清洁 DNA 片段通过限制性消化和凝胶提取获得。通过生物弹射击，产生了 67 个独立的转化体。转化频率达到 3.3%，81%的转基因植物为共转化体。通过Southern 分析证实了 Bt 基因的稳定整合，并且通过Southern 分析确定了插入拷贝数。Western 分析和 ELISA 显示转基因植物中 Bt 蛋白表达水平很高。后代分析证实 Bt 基因根据孟德尔（3:1）比例稳定遗传。昆虫生物测定表明，转基因植物完全免受黄条跳甲的侵害。对 T2 后代植物的 PCR 分析导致高达 4%的无标记转基因水稻植物的恢复。
