Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92204
標題: 柑橘黃龍病潛在分泌性蛋白之分子選殖與分析
Molecular cloning and characterization of the putative secreted effectors of Huanglongbing
作者: 江欣穎
Shin-Ying Jiang
關鍵字: 黃龍病;阿拉伯芥;分泌蛋白;Huanglongbing;Arabidopsis;putative protein
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摘要: 
黃龍病 (Huanglongbing, HLB),是目前已知在柑橘類作物上的一種嚴重病害,其病原菌Candidatus Liberibacter,為革蘭氏陰性菌,主要寄生在宿主篩管細胞內,至今尚無法以人工方式培養,因此無法利用突變及基因重組的方式來進行研究。黃龍病菌透過木蝨 (citrus psyllid)吸食病株韌皮部組織汁液傳播。黃龍病菌感染的植株會出現黃化、葉片不規則黃綠斑塊、小果畸形等徵狀。目前對於黃龍病病原菌的致病機制尚未清楚;經由前人研究預測,得知HLB可編碼出113個分泌蛋白,稱之為Huanglongbing secretion protein, HLBSP。本研究中利用自台中霧峰採集之台灣黃龍病病原菌樣本作為研究材料,共成功增幅29個分泌蛋白基因,稱之為Taiwan Wufeng HLBSP (TWHLBSP)。目前利用生物資訊學以及螢光融合蛋白進行系統性的分析,具有進核特性之分泌性蛋白為TWHLBS026、43、60、72、103等。進一步利用轉基因植物策略,來建構研究黃龍病菌分泌蛋白的研究材料,目前一共建構了27種轉基因擬南芥。初步分析尚未觀察到TWHLBSP轉基因擬南芥與野生型擬南芥有顯著性的差異。另外,利用BLAST分析,發現在Candidatus Liberibacter americanus基因組中,有一蛋白序列與翠菊黃萎病菌質體分泌蛋白SAP11相似,目前命名為CaLaSAP11。本研究同時探討 CaLaSAP11在植物中的功能;透過螢光蛋白的初步分析,發現CaLaSAP11會分布於整個細胞中,但在35S::CaLaSAP11轉基因擬南芥並未觀察到明顯外表型。

Huanglongbing (HLB) is a serious citrus disease caused by the phloem-restricted Gram-negative bacteria, 'Candidatus Liberibacter'. Ca. Liberibacter can be transmitted by the citrus psyllid. The disease symptoms caused by Ca. Liberibacter include yellowish shoots, leaf mottling, small and lopsided fruits. Because Ca. Liberibacter can not be cultured in vitro, the molecular mechanism of HLB disease is largely unknown. Recently, the genome of Ca. Liberibacter has been sequenced and putatively encodes 113 secrection protein. In this study, 29 secretion proteins were successfully amplified from HLB samples collected from Wufeng, Taichung. After bioinformatics and fluorescent fusion protein studies, Taiwan Wufeng HuangLongBing Serection Protein (TWHLBSP) were observed to 5 locate in plant nuclear including TWHLBS026, 043, 060, 072 and 103. Moreover, a total of 27 TWHLBSP teansgenic lines were generated uncover molecular mechanisms of disease symptoms caused by Ca. Liberibacter. The obviously difference have beem not yet observed between the wild-type and TWHLBSP transgenic lines so far. Interestingly, a SAP11 AY-WB homolog CaLaSAP11 was found in Ca. Liberibacter by BLAST analysis. SAP11AY-WB is a secreted protein produced by the phytoplasma causing aster yellows. Here, the subcellular localization of CaLaSAP11 were analyzed and the 35S::CaLaSAP11 transgenic line was generated to study the functions of CaLaSAP11.
URI: http://hdl.handle.net/11455/92204
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