Disease-resistant gene collectin of lectin family of cynoglossus semilaevis
A lectin, a family of technologies, applied in the field of molecular biology, can solve problems such as undiscovered antibacterial functions
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Embodiment 1
[0030] Example 1: Cloning and sequence analysis of the full-length cDNA of the collagen lectin collectin 11 gene of half-smooth tongue sole
[0031] The applicant first screened out a novel lectin family gene collectin 11 with great differential expression through the association analysis (GWAS) of the transcriptome and genome of semi-smooth tongue sole injected with Vibrio anguillarum infection symptoms and those without infection symptoms. The partial mRNA sequence of collectin 11 gene was obtained by horizontal gene amplification, and then the full-length target gene was obtained by RACE-PCR technology, and the full-length cDNA sequence of the collagen lectin 11 gene in half-smooth tongue sole was obtained.
[0032] (1) The sequences of RACE primers and amplification primers used are as follows:
[0033]
[0034]
[0035](2) Experimental method: including Trizol extraction of RNA, cDNA synthesis, PCR amplification, vector construction, transfection of cells, cell cult...
Embodiment 2
[0040] Example 2: Analysis of the expression pattern of the collectin11 gene of half-smooth tongue sole in various tissues of normal fish
[0041] The qRT-PCR primers were designed according to the sequence of collectin11 gene of half-smooth tongue sole. For details of the experimental steps, see the instruction manual of the (SYBR Green) fluorescence quantitative kit, and the qRT-PCR reaction was performed on the ABI-7500Fast real-time quantitative amplification instrument. The qRT-PCR detection results were represented by the mean ± standard error (SE) of three groups of repetitions, the relative expression was calculated using the 2-ΔΔCt method, and the expression histogram was drawn using Origin9.0 ( figure 2 ).
Embodiment 3
[0042] Example 3: Relative expression analysis of the collectin11 gene of tongue sole in half-smooth tongue sole in immune-related tissues after infection with Vibrio vine
[0043] The Vibrio anguillarum infection experiment was carried out on semi-smooth tongue sole, using the Vibrio vine preserved in the laboratory, and the injection dose was 2×10 7 cfu / g, at six time points of 0h, 6h, 12h, 24h, 48h, and 72h after injection, six tissues of liver, spleen, kidney, intestine, gills and blood were collected respectively. Fluorescent quantitative PCR was used to detect the expression of collectin 11 gene in the above six tissues. The primers, reagents and experimental methods used are the same as those described in Example 2.
[0044] The results showed that the expression levels of collectin 11 in various tissues were up-regulated to varying degrees after the injection of Vibrio anguillarum: among them, in the intestine and liver, the expression of collectin 11 was always incre...
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