柑桔潰瘍病原菌致病基因pCL之選殖

Abstract

柑桔潰瘍病病原菌（Xanthomonas campestris pv. citri）是柑桔類植物的致病菌，會造成葉片感染部位的突起，細胞崩解，褐化、木栓化的潰瘍病斑；及水漬狀（water soaking）、黃色暈環（yellow halo）等病徵。移位子誘導的突變株XT-37在病徵表現上僅呈現水漬狀及黃色暈環，而缺少了細胞增生、潰瘍的病徵表現，為callus like minus（CL-）的突變株。 本研究以XT-37（CL-）突變株為對象，分離被Tn5tac1破壞的致病性基因：先建立X. c. pv. citri野生型及突變株XT-37的基因庫；再利用Tn5tac1為標記，由突變株基因庫中篩選出帶有Tn5tacl及部份X. c. pv. citri DNA片段的選殖株。進一步由野生型基因庫中篩選出帶有完整基因的選殖株，將此基因命名為pCL基因。而後定出此基因的限制?圖譜與部份DNA序列，以互補實驗定出基因之最小範圍。此外，經由限制?剪切與南方轉印法觀察到，pCL基因位於X. c. pv. citri的質體上。研究中並發現，尚有其他3段DNA與pCL基因有很高的相似性，同時也位於X. c. pv. citri質體上。另外由XT-37的性狀分析，推測被破壞的基因可能與IAA有關，但是經IAA生物分析（IAA bioassay），發現在L-tryptophan的存在下，IAA的產量在突變株XT-37與野生型菌株中並無明顯差異，顯示XT-37的病徵可能還有其他因數影響。

Xanthomonas campestris pv. citri is a plant pathogenic bacterium, which causes citrus canker on citrus plants. Symptoms of citrus canker disease include erumpent, corky lesions, water soaking and yellow halos surrounding the infected area. The Tn5tac1-induced pathogenic mutant, XT-37, which showed water soaking dark spots and surrounded yellow halos on the host, but lost the ability of forming canker-like lesions. Therefore, this gene that transposon tagging in XT-37 is named pCL (pathogenic gene for canker like). To isolate the pCL gene, genomic libraries of wild type X.c.pv.citri and XT-37 were constructed. Using restriction mapping, Southern blotting and hybridization techniques, the insertion site of transposon and the restriction map of putative pCL gene was established. Except the pCL gene, three additional homologous fragments were found, and all of them located on a indigenous plasmid of X.c.pv.citri. On the other hand, base on the symptoms of XT-37 that developed only water soaking and no callus forming, the production of auxin was suggested to be investigated. However, there is no significant difference of IAA production between the wild type strain and the XT-37 mutant.