Development of super-Agrobacterium for the efficient production of transgenic plants in Cucurbitacae

本研究的目的是：1）阐明植物-农杆菌间通过乙烯信号途径的相互作用; 2）将农杆菌分子育种的知识应用于葫芦科植物，以提高农杆菌的基因转移能力。主要结果如下：1.我们观察到乙烯本身并不抑制农杆菌细胞的生长，而植物对乙烯的反应是抑制农杆菌vir基因在植物细胞上的表达。这种抑制作用可能是由植物细胞中乙烯诱导/抑制的物质引起的。这些结果使我们推测，我们可以开发超级农杆菌，显示更高的基因转移能力，通过提供抑制乙烯生物合成的植物细胞中的能力.根瘤毒素是大豆根瘤菌（Bradyrhizobium elkanii）产生的一种乙烯生物合成抑制剂，可抑制寄主植物的乙烯合成。来自B的rtxACDEFG基因。elkanii和OAH合酶基因导入根癌农杆菌C58菌株，获得了高产根瘤毒素的基因工程菌。ACC脱氨酶是由共生细菌产生的一种酶，它降解ACC，从而抑制植物体内乙烯的生物合成。将假单胞菌ACC脱氨酶基因导入A.根癌农杆菌C58菌株，转基因农杆菌具有ACC脱氨酶活性.转基因农杆菌无论是产生根瘤毒素还是ACC脱氨酶，都表现出较高的转基因能力。此外，ACC脱氨酶产生菌对拟南芥的转基因能力也较强。总之，我们成功地通过分子育种获得了对植物细胞具有较高转基因能力而不抑制vir基因表达的超级农杆菌。

The objectives of the research were 1) to elucidate the plant-Agrobacterium interaction through ethylene signaling pathway and 2) to apply the knowledge on molecular breeding of Agrobacterium which shows higher ability of gene transfer to Cucurbitacae plants. The followings are the major results obtained.1. We observed that ethylene itself did not inhibit Agrobacterium cell growth whereas plants responding to ethylene, that inhibiting the vir gene expression in Agrobacterium on the plant cells. It is likely that the inhibitory effect is caused by materials which are induced/depressed by ethylene in the plant cells. These results allow us to speculate that we can develop super-Agrobacterium which shows higher ability of gene transfer by providing the ability to inhibit ethylene biosynthesis in plant cells.2. Rhizobitoxine is an ethylene biosynthesis inhibitor produced by soybean root nodule bacteria (Bradyrhizobium elkanii), and reduces ethylene production from the host plants. rtxACDEFG genes from B. elkanii and OAH synthase gene from Mesorhizobium loti were introduced to Agrobacterium tumefaciens C58 strain, and the genetically engineered Agrobaterium produced rhizobitoxine. ACC deaminase is an enzyme produced by symbiotic bacteria, and degrade ACC which results in inhibition of ethylene biosynthesis in plants. ACC deaminase gene from Pseudomonas strain was introduced to A. tumefaciens C58 strain, and the genetically engineered Agrobacterium showed ACC deaminase activity.3. The genetically modified Agrobacterium producing either rhizobitoxineor ACC deaminase showed higher ability of gene transfer to melon. In addition, the Agrobacterium producing ACC deaminase also showed higher ability of gene transfer to Arabidopsis.In conclusion, we are succeeded in molecular breeding to production of so-called super-Agrobacterium which shows higher ability of gene transfer into plant cells without inhibition of vir gene expression.