A dsf degrading enzyme coding gene fady and its application
A technology encoding gene and degrading enzyme is applied to the DSF degrading enzyme encoding gene fadY and its application field, which can solve the problems of affecting the degradation ability, not being a degradation gene, and failing to find a degradation gene, so as to improve disease resistance and wide application prospects. Effect
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Embodiment 1
[0033] Whole genome sequencing analysis of embodiment 1 Acinetobacter lactucae (Acinetobacter lactucae) strain QL-1
[0034] In recent years, with the rapid development of high-throughput sequencing technology, bioinformatics has gradually become an important tool for auxiliary research in various fields. Using the genome information of sequenced strains and some open database platforms, functional gene annotation and positioning are carried out, and their possible involvement This is an effective strategy to obtain genes with related functions. In this example, the whole genome sequencing method was used to search for possible degradation genes in the strain QL-1 through the analysis of the genome characteristics of the strain QL-1 and the results of functional gene annotation. The specific experimental methods and results are as follows:
[0035] (1) Genomic characteristics of strain QL-1
[0036] In order to understand the gene function and structure of strain QL-1, the w...
Embodiment 2
[0040] Example 2 Obtaining of DSF degrading enzyme encoding gene fadY
[0041] In this example, according to the results of genomic feature analysis and functional gene annotation of the strain QL-1 in Example 1, possible degradative enzyme gene mutants were constructed for verification to find highly efficient degradative enzymes. The specific experimental methods and results are as follows:
[0042] (1) Experimental method
[0043] According to reports, the rpfB gene located upstream of the DSF synthesis gene rpfF in Xanthomonas campestris Xcc was identified as an acyl-CoA ligase encoding gene, which is involved in the degradation of DSF family signaling molecules and regulates the expression of the QS system.
[0044] Through the results of the whole genome sequencing analysis of the bacterial strain QL-1 in Example 1, genes related to fatty acid degradation were found in the whole genome of the bacterial strain QL-1, and these genes were compared with the rpfB gene by BLA...
Embodiment 3
[0049] Example 3 Optimization, expression and purification of DSF degrading enzyme FadY
[0050] (1) Experimental method
[0051] According to the results of the whole genome sequencing analysis of Example 1 Acinetobacter lactucae (Acinetobacter lactucae) strain QL-1, gene fadY is speculated to encode a long-chain fatty acyl-CoA ligase, encoding 559 amino acid (aa) proteins, and the predicted molecular weight is 61.53kDa, the isoelectric point is 8.03.
[0052] In order to obtain a higher yield and a purer degrading enzyme, the codon optimization of the gene fadY was carried out. The codon-optimized DNA fragment encoding DSF degrading enzyme FadY was amplified by PCR, and subcloned into the expression vector pGEX-6p-1 to obtain the recombinant plasmid pGEX-fadY, which was transformed into Escherichia coli BL21 to obtain the recombinant Bacteria E.coil BL21 / pGEX-fadY. The bacteria were inoculated in LB medium and cultured at 37°C, and 100 mg / mL of ampicillin was added, when ...
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