柑桔潰瘍病原菌引起柑桔類植物水漬狀病徵基因之分析

Abstract

柑桔潰瘍病原菌(Xanthomonas axonopodis pv. citri)主要感染柑桔類植物的葉片和果實，在感染的部位造成細胞增生，周圍並形成水漬狀病斑。以移位子(transposon) Tn5tac1誘導的突變株XT27，在感染寄主後僅在葉片上造成類似癒合組織的病徵，未引起水漬狀病斑，為water soaking minus (WS-)的突變株。本研究以XT27 (WS-)突變株為對象，分離被Tn5tac1破壞之致病性基因：以Tn5tac1 flanking DNA為探針，由野生型菌株基因庫中選殖(screen)出一菌株HB101 (pHW27)，經由南方轉印法(southern blotting)及雜合反應(hybridization)，確認此菌株帶有移位子旁的野生型菌株染色體DNA片段，並進行被Tn5tac1破壞的基因附近之限制?圖譜分析，之後進行互補實驗，確認當初被破壞的基因之範圍。將帶有此片段的質體pUW27NotE5.1以電導法導入XT27突變株，能使XT27突變株在檸檬葉片上恢復與野生型菌株所產生的病徵相同，即有類似癒合組織突起並有水漬狀病斑出現，因此認為Not I-Eco RI 5.1 kb DNA片段已包含當初被破壞的基因，將此片段輸入原核生物資料庫比對，推測此片段包含有glnA基因、glnB基因與amtB基因。glnA基因負責合成麩氨醯胺合成?，glnB基因負責合成P-Ⅱ調控蛋白，amtB基因負責合成銨離子轉運蛋白，這三個基因的產物皆與麩氨醯胺(glutamine)的生合成有關。因此，推測麩氨醯胺的合成，可能和柑桔潰瘍病原菌所造成的水漬狀病徵有直接或間接之相關性。

Xanthomonas axonopodis pathovar (pv.) citri, a plant pathogenic bacterium, mainly infects citrus to cause citrus canker. The wild type, XW47, makes symptoms of citrus canker, including erumpent, corky lesions, water soaking and yellow halos surrounding the infected area. Transposon Tn5tacl inserted into chromosome of X. axonopodis pv. citri, and induced a random mutagenesis to generating the mutant XT27. After infection, the mutant, termed mutant WS-, resulted in a callus-like and an erumpent lesion, which are different from the wild type. The purpose of this study is to isolate the infection gene of XT27 (mutant WS-) destroyed by transposon Tn5tac1. First, the probe of Tn5tac1 flanking DNA was used to select HB101 (pHW27) from the XW47 genomic library. HB101 (pHW27), which contained wild type DNA, was documented by methods of southern blotting and hybridization. Then, the complementation analysis revealed the destroyed genes are included in the DNA fragment of Not I-Eco RI 5.1 kb. Finally, sequence analysis reveals that the DNA fragment exists genes glnA, glnB and amtB. These genes glnA, glnB and amtB, encode glutamine synthetase, nitrogen P-Ⅱ regulatory protein and ammonium transporter, respectively. These products are relating to the biosysthesis of glutamine. It is supposed that the biosysthesis of glutamine is possible directly or indirectly correlating to the symptom of water soaking caused by X. axonopodis pv. citri.