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首页> 外文期刊>Journal of bacteriology >Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli.
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Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli.

机译：菊花欧文氏菌果胶裂解酶基因的分子克隆及其在大肠杆菌中的表达。

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A genomic library of Erwinia chrysanthemi EC16 was constructed in plasmid pHC79, and seven putative pectate lyase (PL) clones in Escherichia coli were selected on pectate agar. Six of the recombinant cosmids contained a common PstI fragment of ca. 8.2 kilobases (kb). Subcloning of this fragment in either orientation into the PstI site of plasmid pBR329 resulted in E. coli transformants that produced a PL of pI 9.8 which was indistinguishable from one of two PLs produced by strain EC16. A 6.6-kilobase PstI fragment from the remaining cosmid clone caused production of an Erwinia PL of pI 8.8 when the fragment was subcloned in either orientation into plasmid pBR329 and transformed into E. coli. Selected pBR329 subclones for the 8.2- and 6.6-kilobase PstI fragments showed no similarity in their restriction maps and did not cross-hybridize. All of the E. coli cosmid clones that produced large amounts of PL also caused soft-rot of potato tubers and tuber slices, thus confirming the role of the enzymes in plant tissue maceration. The E. coli cosmid clones and plasmid pBR329 subclones produced the PLs constitutively, unlike Erwinia chrysanthemi, which made the enzymes inducibly. However, catabolite repression appeared to function in the E. coli clones, and almost all of the PL activity occurred in the periplasm and culture fluids. Thus, the Erwinia PL clones appear to contain signal peptide sequences, transcription and translation signals, and a recognition sequence for the catabolite activator protein, all of which function efficiently in E. coli.
机译：在质粒pHC79中构建了菊花欧文氏菌EC16的基因组文库，并在果胶琼脂上选择了大肠杆菌中的七个假定的果胶酸裂合酶（PL）克隆。六个重组粘粒包含一个大约一个的共同PstI片段。 8.2千米（kb）。将该片段以任一方向亚克隆到质粒pBR329的PstI位点中，产生了大肠杆菌转化体，其产生了pI 9.8的PL，其与菌株EC16产生的两个PL之一没有区别。当将该片段以任一方向亚克隆到质粒pBR329中并转化到大肠杆菌中时，来自剩余粘粒克隆的6.6-碱基碱基的PstI片段导致产生pI 8.8的欧文氏菌属PL。为8.2和6.6碱基碱基的PstI片段选择的pBR329亚克隆在其限制性酶切图中没有显示相似性，也没有交叉杂交。所有产生大量PL的大肠杆菌粘粒克隆也引起马铃薯块茎和块茎切片的软腐，从而证实了酶在植物组织浸软中的作用。大肠杆菌粘粒克隆和质粒pBR329亚克隆是组成型产生PL的，而不同于欧文氏菊（Erwinia chrysanthemi），后者可诱导产生酶。但是，分解代谢物阻遏似乎在大肠杆菌克隆中起作用，几乎所有的PL活性都发生在周质和培养液中。因此，欧文氏菌属PL克隆似乎包含信号肽序列，转录和翻译信号以及分解代谢物激活蛋白的识别序列，所有这些在大肠杆菌中均有效发挥功能。

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