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

本研究的目的是：1)通过乙烯信号通路阐明植物与农杆菌的相互作用；2)将具有较高基因转移能力的农杆菌的分子育种知识应用于葫芦科植物。以下是获得的主要结果。我们观察到乙烯本身不抑制农杆菌细胞的生长，而植物对乙烯的反应抑制了农杆菌中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，通过提供抑制植物细胞中乙烯生物合成的能力，我们可以开发出具有更高基因转移能力的超级农杆菌。根瘤素是一种由大豆根瘤菌（elkanii缓生根瘤菌）产生的乙烯生物合成抑制剂，可减少寄主植物的乙烯产量。将elkanii的rtxACDEFG基因和loti中根瘤菌的OAH合成酶基因导入农杆菌C58菌株，经基因改造的农杆菌产生根瘤菌毒素。ACC脱氨酶是一种由共生细菌产生的酶，能够降解ACC，从而抑制植物的乙烯合成。将假单胞菌ACC脱氨酶基因导入到农杆菌C58中，经基因改造后的农杆菌具有ACC脱氨酶活性。产生根霉毒素或ACC脱氨酶的转基因农杆菌转移到甜瓜上的能力较高。此外，产生ACC脱氨酶的农杆菌也表现出较高的基因向拟南芥转移的能力。总之，我们成功地进行了分子育种，产生了具有较高的基因转移到植物细胞的能力而不抑制vir基因表达的所谓超级农杆菌。

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.