Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals
Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals
复制标题
DOI：
10.1038/83559
复制
发表时间：
2001-01-01
影响因子：
46.9
通讯作者：
Daniell, H
中科院分区：
文献类型：
--
作者：
De Cosa, B;Moar, W;Daniell, H
在核转基因植物中，多个基因的表达需要引入单个基因和耗时的后续回交以重建多亚基蛋白或途径，这是一个因可变表达水平而复杂的问题。为了在单个转化事件中实现多个基因的表达，我们已经将几个基因引入到质体基因组中。我们通过PCR和Southern印迹分析证实了cry 2Aa 2操纵子在T-0和T-1转基因植物中的稳定整合。外源蛋白在成熟叶片中积累占总可溶性蛋白的45.3%，即使在老的漂白叶片中也保持稳定（46.1%），从而提高了转基因植物在整个生长季节的功效和安全性。这代表了迄今为止在转基因植物中报道的外源基因表达的最高水平。通常难以控制的昆虫（10天大的棉铃虫、甜菜夜蛾）在食用转基因叶片后被100%杀死。电子显微照片显示存在折叠成立方晶体的杀虫蛋白。外源蛋白晶体的形成（由于推定的伴侣蛋白ORF 2的过表达和折叠）提供了一种简单的离心纯化方法，并通过保护免受细胞蛋白酶的影响提高了稳定性。操纵子在转基因植物中表达的证明为在单个转化事件中在植物中工程化新途径铺平了道路。
In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T-0 and T-1 transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.