火鶴花細菌性葉桔病菌新插入序列ISXCD1之分離與分析

Isolation and Characterization of a New Insertion Sequence, ISXCD1, from Xanthomonas axonopodis pv. dieffenbachiae, the Causal Agent of Anthurium Blight

李嘉榮(Lee, C. J.) ； 劉秀玲(Liu, H. L.) ； 林長平(Lin, C. P)

《植物病理學會刊》 13卷3期 (2004/09) Pp. 219-232

https://doi.org/10.6649/PPB.200409_13(3).0007

摘要

在本研究中，成功地使用 pUCD800 質體誘釣獲得火鶴花細菌性葉枯病菌 Xonthomooas axonopodis pv. diffenbachiae（簡稱XAD之一新插入序列(insertion se quence , IS )。實驗中使用電穿孔法（electroporation）將pUCD80O質體導入XAD 中，由於質體上sacBR基因所表現之evansucrase，致使成功導入具完整sacBR質體之葉枯病菌轉形株（transformant )，無法生長於含有蔗糖之培養基。實驗中，利用轉形株在蔗糖培養基上培養特性之改變，可篩選，acsBR基因被轉位子插壞者，並對其內之pUCD800進行質體分析，經內鑑識酵素 EcoRI及HindIII酵解後，分析其圖譜並觀察sacBR區域變化。總計篩選了186株突變株，其中 71 株為質體大小完全不變者，28株在，sacBR區域有明顯插1.2 kb片段，87株質體大小發生不可預期的重組現象。依PCR測定及序列分析結果顯示，此，sacBR區域中之1.2kb插入片段之28株中有21株為插入序列IS1051所引起之插入點不活化，另7株則為屬於IS3 family 之新插入序列，此一新插入序列已正式登錄並命名為ISXCDI，其全長為1,203bp，包括兩端 38 bp 的不完全（imperfect）反向重複序列（inverted sequence, IR)，其中包含有11 bp的錯配 (mismatchs )，在轉形株15- 4及27-25之，sacBR基因上出現4bp同向重複序列（direct repeat , DR)，對應肢基酸序列後發現其為兩個有部分重疊的open reading frame (ORF) : orfA及orfB，在重疊區域可發現 IS3 framily轉位酶常見的A(下標7)T motif，在 ORFA也可找到helix-tum-helix motif、ORFB也可找到保守的DDE motif ，其均為IS3 family轉位醇之共通特性。由南方氏雜配分析得知ISXCD1在XAD基因組中有9- 11個雜配訊號，並且也廣泛存在於親緣較近之xanthomonas spp. 細菌中。利用反向聚合酵素連鎖反應（inverse polymerase chain reaction , IPCR )，找到此插入序列在基因組中獨立的五個位置，比對此些插入序列周圍之序列，並未發現有明顯之插入點偏好性。

關鍵字

火鶴花細菌性葉枯病 ； 插入序列 ； 反向聚合酵素連鎖反應

並列摘要

A new insertion sequence, ISXCD1, is successfully isolated from Xanthonzonas axonopodis pv. dieffenbachiae (XAD) by disrupting the sacBR-harboring plasmid, pUCD800, which was introduced into XAD via electroporation. Based on restriction enzyme analyses, the plasmids isolated from 186 sucrose- resistant colonies are divided into three classes: insertions in the sacBR element of pUCD800 (18.8%), no noticeable change in the structure of pUCD800 (38.2%), and rearrangement of pUCD800 (46.77%). ISXCD1 (accession no. AF263433) is identified from the insertions of the sacBR element. ISXCD] is 1,203 bp in size, contains 38 bp inverted repeats with 11 bp mismatches at its termini, and carries two overlapping open reading frames (orfA and orfB) with an A7T motif in the overlapping region. It generated 4-bp target site duplication after transposition. The deduced amino acid sequences of orfA and orfB contained a potential helix-turn-helix motif and a DDE domain of transposases, respectively. ISXCD] is present about 9-11 copies in the XAD genome according to the results of Southern hybridization. Southern hybridization analyses also reveal ISXCDJ is widely distributed in G(-)and G(+)bacteria. Sequence analysis of five IPCR products obtained from various ISXCD1-inserted locations of genome indicated there is no preferred target sequence for its transposition.

並列關鍵字

anthurium blight ； inserton sequence ； IS ； inverse polymerase chain reaction ； IPCR