Erwinia carotovora subsp. carotovora低分子量細菌素選殖與表現

Abstract

Erwinia carotovora subsp. carotovor (E.c.c.)常造成植物的軟腐病，目前為止對於此菌種所造成的細菌性軟腐病的防治仍找不到有效的方法。此研究的目標為找到一種廣泛且有效力抑制E.c.c.生長的細菌素，希望能以非農藥方式，達成軟腐菌對植物病害防治。針對本實驗室以及本校植病所曾國欽老師實驗室所保存的E.c.c.，進行細菌素活性調查，發現本實驗室所保存的3F-3菌株所分泌的細菌素具有高度的抑菌活性。 首先利用跳躍因子Tn5在染色體上進行插入突變，阻斷3F-3菌株低分子量細菌素產生後，我們利用TAIL-PCR的方法增幅並定序各個突變菌株被Tn5阻斷的周圍序列，經由美國國家衛生研究院醫學圖書館的資料庫(NCBI blast)以DNA或胺基酸序列比對，結果發現突變株TF1-2所阻斷位置為細菌素生產基因，此段基因是未曾被發現過的新規細菌素基因，因此我們把它命名為carocin S2。 由3F-3 菌株的genomic DNA分離出長度為5705bp的片段，它包含6個open reading frames (ORFs), 其中ORF2轉譯後胺基酸序列與colicin D activity蛋白有38%的同質性，colicin D屬於核醣核酸水解酶; ORF3轉譯後胺基酸序列與colicin D immunity 蛋白有32%的同質性，colicin D immunity蛋白的作用是抑制colicin D activity蛋白活性保護宿主細胞。因此我們將ORF2命名為caroS2K而ORF3命名為caroS2I，這兩段基因合稱為carocin S2。將此DNA片段連接於pBR3222 和pMCL210質體建構出pBR322-119 和pMCL210-119以轉形作用分別送入DH5α、Ea1068以及TF1-2三個沒有生產細菌素能力的菌株，實驗發現帶有pMCL210-119質體的菌株均能表現出低分子量細菌素活性，因此我們成功的將E.c.c.低分子量細菌素基因選殖並表現出它的抗生效果。

The Erwinia carotovora subsp. carotovovra (E.c.c.) causes soft-rot disease of many plant species. Despite its economic importance, no efficient method has been found to control this disease. The bacterium produces the antibacterial substances called bacteriocins owning the ability to identify the similar bacteria and kills them. We want to find out the bacteriocin of E.c.c. being able to kill most of subspecies, and use the biological control this disease. By bacterial mating experiment a transposon Tn5 insertion mutant TF1-2 was obtained, which lost the low-molecular-weight bacteriocin(LMWB) producing ability. After the first TAIL-PCR experiment, the PCR products were isolated and the DNA sequences were determined from the Tn5 insertion junction site. The DNA probe was prepared from TAIL-PCR result and it was used in Southern hybridization experiments. A 5705bp fragment was isolated from 3F-3 DNA library, and it contains six open reading frames (ORFs). The ORF2 encodes a protein 38% identical to colicin D activity protein, which is ribonuclease activity. The ORF3 encodes a protein 32% identical to immunity protein of colicin D, which inhibition the bacteriocin activity to host cell. It was therefore proposed that ORF2 be designated as caroS2K, and ORF3 as caroS2I. The two genes were named carocin S2 gene. We cloned the carocin S2 gene into pBR322 or pMCL210 vector, and the expression plasmids pBR322-119 and pMCL210-119 were obtained. After pBR322-119 or pMCL210-119 into E. coli DH5α, Ea1068, or TF1-2 cells, the results showed that DH5α/pMCL210-119, Ea1068/pMCL210-119 and TF1-2/pMCL210-119 can produce the low-molecular-weight bacteriocin by bacteriocin assay experiments. This result suggests that the carocin S2 gene is a LMWB genes contain a killing protein and an immunity protein.