Vanjildorj Enkhchimeg, Song Seo Young, Yang Zhi Hong, Choi Jae Eul, Noh Yoo Sun, Park Suhyoung, Lim Woo Jin, Cho Kye Man, Yun Han Dae, Lim Yong Pyo
Department of Horticulture, Chungnam National University, Gung-dong, Yuseong-gu, Daejeon 305-764, Korea.
Plant Cell Rep. 2009 Oct;28(10):1581-91. doi: 10.1007/s00299-009-0757-4. Epub 2009 Aug 14.
We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2-3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.
我们培育出了一种对软腐病具有高耐受性的转基因大白菜（Brassica rapa L. ssp. pekinensis）自交系“Kenshin”。通过高效的农杆菌介导转化方法，在大白菜中表达N-酰基高丝氨酸内酯酶（AHL-内酯酶）赋予了其耐受性。为了在大白菜中合成并表达AHL-内酯酶，将与PinII信号肽（来自马铃薯的蛋白酶抑制剂II）融合的aii基因（来自芽孢杆菌属GH02的AHL-内酯酶基因）导入植株。通过在含潮霉素的培养基上生长筛选出了5个转基因株系（转化效率为3.7%）。Southern杂交分析表明转基因已稳定整合到基因组中。在这5个转基因株系中，4个株系观察到单拷贝整合，1个转基因株系观察到双拷贝整合。Northern杂交分析证实pinIISP-aii融合基因在所有转基因株系中均有表达。在组织和幼苗阶段对软腐病耐受性进行了评估。与野生型植株相比，转基因植株对软腐病的耐受性显著增强（2至3倍）。因此，融合基因pinIISP-aii的表达降低了大白菜对软腐病的易感性。我们得出结论，由aii编码的重组AHL-内酯酶能够有效淬灭细菌群体感应并预防细菌群体密度依赖性感染。据我们所知，本研究首次证明了大白菜自交系“Kenshin”的转化，并且首次描述了融合基因pinIISP-aii对增强软腐病耐受性的作用。
