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標題: Mutational studies of xpsD gene of Xanthomonas campestris pv. campestris
作者: 陳戴瑜
Chen, Day-Yu
關鍵字: Xanthomonas campestris pv. campestris;十字花科黑腐病菌;extracellular enzyme;胞外酵素;干擾分泌
出版社: 分子生物研究所
XpsD 為一外胞膜酯蛋白,是十字花科黑腐病菌分泌胞外酵素所必需的蛋
白質之一。本研究利用一系列 xpsD 變異基因,探討 XpsD蛋白在胞外酵
素分泌過程中可能的功能。以西方墨點法檢查 XpsD在細胞內的分佈位置
,結果顯示 XpsD變異蛋白之分佈與野生株相同,免疫螢光標定實驗亦顯
示,各變異蛋白均嵌入外胞膜,並有部分裸露於菌體外。除 pCD103及
pKU2 合成的 XpsD變異蛋白具分泌功能外,其餘皆喪失分泌功能。將變異
基因以廣寄主性載體送入野生株 XC1701,利用固體培養基檢查澱粉及
的現象,包括 pCD105,pKD2,pKdA6 及 pYL4 所合成的 XpsD 變異蛋白
;pMH7,pKdPs 及 pKD20 所表現者,則無顯著干擾。利用澱粉抗體檢
查XC1701轉接合菌株之澱粉分泌情形時, pMH7,pKdPs 及 pKD20表現
的 XpsD 變異蛋白亦呈現微弱的干擾現象。以 XpsD 抗體偵測 xpsD基因
表現時,除成熟型之 77kDa XpsD 蛋白產物外,尚有一分子量約為55kDa
之XpsD55 的產物出現,為探討 XpsD55是否為 xpsD基因中連續 6 個 A
經 translational frameshift 後,提早終止所產生。本研究第二部分將
連續6個 A 序列改換為 CAAGAA序列,並進行 6 個 A 序列的刪除,發現
對 XpsD55 的產生沒有影響;同時於6個 A 之上、下游構築 xpsD-phoA
融合基因的實驗結果,亦證實 XpsD55的出現,可能與 6 個 A 造成
frameshift 的關聯性不大。
XpsD is an outer membrane lipoprotein, required for the
secretion of extracellular enzymes by Xanthomonas campestris
pv. campestris. A series of mutant xpsD genes were constructed.
Immunodetection of fractionated cell extracts using antibody
against XpsD showed that the mutant XpsD proteins were, similar
to the wild type XpsD, mainly located in membrane. Immunofluo-
rescence labeling in intact cells further revealed that some
regions of the proteins were exposed to the cell surface.
Results from complementation experiments indicated that all
mutants, except two (encoded in pCD103 and pKU2), were
defective in protein secretion. Secretion interference was
caused by introducing into XC1701 some (encoded in pCD105,
pKD2, pKdA6, pYL4), but not the other (encoded in pMH7, pKdPs
and pKD20) nonfunctional, mutant xpsD genes. Cell fractionation
followed by immunodetection using antibody against α-amylase
revealed that slight interference in secretion was also caused
by mutant XpsD made from pMH7, pKdPs, and pKD20. The molecular
weight of mature XpsD is 77kDa. In addition to the 77kDa
product, a 55kDa protein, XpsD55, could also be detected on
immunoblot. In order to study the significance of the A6T
sequence in xpsD coding region in the production of XpsD55,
base substitutions (CAAGAA), A6T deletion and XpsD-PhoA fusions
were constructed. The results suggested that XpsD55 was
probably not produced as a result of frameshifting downstream
of A6T.
Appears in Collections:分子生物學研究所

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