Pelteobagrus fulvidraco red head disease resistance character related SNP marker, screening method and application
A screening method, a technology for yellow catfish, applied in the fields of biochemical equipment and methods, recombinant DNA technology, determination/inspection of microorganisms, etc.
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Embodiment 1
[0039] 1. Screening of CypA gene structure and SNP sites of yellow catfish
[0040] The screening of SNP sites in the CypA gene is mainly composed of two parts: preliminary prediction of DNA pool sequencing and individual verification. The specific steps are as follows:
[0041] (1) Before the challenge experiment of yellow catfish, randomly select 30 male yellow catfish selected from Xiantao City, Hubei Province, cut off the caudal fin, use the phenol-chloroform method to extract the genomic DNA of the fin ray sample, and detect the extracted the quality of the DNA.
[0042](2) After mixing equal amounts of genomic DNA from 30 individuals, send it to Beijing Novogene Technology Co., Ltd. for library construction and sequencing. Among them, the qualified library was sequenced on the Illumina HiSeq-PE150 platform. After getting off the plane, perform quality control on the off-plane data, filter the low-quality data, and obtain high-quality data (Clean Data).
[0043] (3) Us...
Embodiment 2
[0077] Example 2 Transgenic zebrafish strain challenge experiment
[0078] 1. Construction of transgenic zebrafish strains expressing CypA of peltatus catfish
[0079] (1) Construction of expression plasmid
[0080] 1) Extract the yellow catfish RNA and reverse transcribe it into cDNA through the kit for future use.
[0081] 2) Obtain the ORF sequence of CypA of yellow catfish from NCBI, use Primer 5.0 software to design primers, and introduce XmaI restriction sites into the upstream primer and downstream primer respectively.
[0082] CypA ORF amplification primers
[0083]
[0084] 1) PCR amplification reaction system is the same as above
[0085] 2) PCR amplification reaction conditions are as follows:
[0086]
[0087] 3) Amplify the target band by PCR, and recover the PCR product with a kit;
[0088] 4) Digest the PCR product and the pTol2-EGFP vector with XmaI respectively, and purify and recover the digested products;
[0089]
[0090] The above enzyme dig...
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