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.

CN111662987AInactive Publication Date: 2020-09-15HUAZHONG AGRI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2020-09-15
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a pelteobagrus fulvidraco red head disease resistance character related SNP marker, a screening method and an application. A CypA gene is analyzed based on pelteobagrus fulvidraco genome information and a mixed pool sequencing technology to obtain a potential SNP locus, and primer amplification is designed for verification. Association analysis is carried out on the potential SNP loci and the red head disease of the pelteobagrus fulvidraco to find that one SNP has significant difference, and allele mutation (T / C) exists on the 528th nucleotide of the CypA genome sequence of the SNP. The SNP 528T / C also has significant difference in the pelteobagrus fulvidraco of another culture group. Besides, a transgeniczebra fish line expressing the yellow pelteobagrus fulvidraco CypA is constructed, and toxicity attacking experiments prove that the transgenic zebra fish has better resistance to Edwardsiella ictaluri, and further illustrates the true effectiveness of the correlation between the SNP site of the pelteobagrus fulvidraco CypA gene and the pelteobagrus fulvidraco ''red head disease'' resistance.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP marker, a screening method and an application related to anti-red head disease traits of yellow catfish. Background technique

[0002] Pelteobagrus fulvidraco belongs to the Siluriformes, Pelteidae, Pelteobagrus genus, and is one of the important freshwater economic fishes in my country. In recent years, Chinese researchers have carried out a lot of research on the reproductive regulation and species improvement of the yellow catfish. The successive appearance of the new species of yellow catfish all-male yellow catfish "Quanxiong No. 1" and the hybrid yellow catfish "Huangyou No. 1" has promoted the development of the yellow catfish industry. However, due to factors such as high-density intensive aquaculture and water environment pollution, various diseases of the yellow catfish broke out and farmers suffered heavy losses. Common diseases of yellow catfish includ...

Examples

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...