cSNP molecular marker detection kit and method for scylla paramamosain disease-resistant characters

A technology of pseudo-cave blue crabs and molecular markers, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., and can solve the problems of heavy workload and poor application effect of molecular markers related to disease resistance

Active Publication Date: 2017-06-09
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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AI Technical Summary

Problems solved by technology

[0005] At present, there are few detection indicators related to the breeding traits of mud crab disease resistance, and the establishmen...

Method used

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  • cSNP molecular marker detection kit and method for scylla paramamosain disease-resistant characters
  • cSNP molecular marker detection kit and method for scylla paramamosain disease-resistant characters
  • cSNP molecular marker detection kit and method for scylla paramamosain disease-resistant characters

Examples

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Embodiment 1

[0070] Example 1: Acquisition of the full-length nucleic acid sequence and amino acid sequence of the disease resistance factor SpALF6 of Scylla pseudomae

[0071] Total RNA was extracted from the blood cell samples of Scylla pseudocarpus, and after testing to meet the requirements, the cDNA sequences of a large number of genes were obtained through transcriptome sequencing and data analysis. Using the online software blastx to analyze one of the full-length 756bp sequences, it was found that the sequence was 78% homologous to Portunus trituberculatus PtALF6 in the Genbank database, indicating that the sequence was a new member of the ALF family of Scylla cryptae, named SpALF6, its nucleotide sequence and predicted amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0072] Among them, the full-length cDNA of SpALF6 (SEQ ID NO: 1) contains 681 bp, wherein the 79th to 426th nucleotides are the open reading frame (SEQ ID NO: 5) encoding the amino acid ...

Embodiment 2

[0074] Example 2: Acquisition of SpALF6 single nucleotide mutant SpALF6-M sequence

[0075] The DNA sequence of the single nucleotide mutant SpALF6-M was obtained by fusion PCR technology. The primers SpALF6EF:5'-TACTCAGAATTCTTCGTGAAGGAACTACTCT-3'(SEQ ID NO:6) and SpALF6MR:5'-TACTCGGTAGTTACAGACACGGTC-3'(SEQ ID NO:8) were paired, and the mutation site was obtained by PCR using the SpALF6 nucleic acid sequence as a template (Containing) the upstream nucleic acid fragment; SpALF6MF:5'-TCCTTGACCGTGTCTGTAACTAC-3'(SEQ ID NO:9) and primer SpALF6ER:5'-TACTCACTCGAGCTATTCTAAGAAGAGTCG-3'(SEQ ID NO:7) are paired, using the SpALF6 nucleic acid sequence as a template, Obtain nucleic acid fragments downstream of the mutation site (including) by PCR; take 0.5 μL of the above two PCR products, mix them as subsequent PCR templates, and use SpALF6EF and SpALF6ER as paired primers to obtain the corresponding SpALF6-M mature peptide region by PCR. nucleic acid sequence. Finally, compared with th...

Embodiment 3

[0076] Embodiment 3: Construction of SpALF6 and SpALF6-M prokaryotic expression vector

[0077] The DNA sequence corresponding to the SpALF6 mature peptide was amplified with the upstream primer SpALF6EF and the downstream primer SpALF6ER. The PCR reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 53°C for 45 s, extension at 72°C for 45 s, a total of 35 cycles, and a final extension at 72°C for 5 min. PCR products were analyzed by 1.5% agarose gel electrophoresis and positive fragments were recovered.

[0078] The above-mentioned purified and recovered nucleic acid fragments and the expression vector pET30a were respectively digested with endonucleases EcoRI and XhoI, and after digesting at 37°C for 3 hours, 1 μL of the digested products were subjected to 1% agarose gel electrophoresis to detect the digestion effect. The electrophoresis results of the purified and recovered nucleic acid fragments and the expression vector...

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Abstract

The present invention relates to a cSNP molecular marker detection kit and a detection method for scylla paramamosain disease-resistant characters, relates to the technical field of molecule auxiliary disease resistance breeding in aquatic livestock, and particularly relates to a single nucleotide polymorphism cSNP molecular marker, capable of influencing disease resistance of blue crabs, in a cDNA sequence of scylla paramamosain SpALF6, as well as a detection kit and a detection method thereof. On a basis that a disease resistance factor SpALF6 of the scylla paramamosain is identified, by performing sequence comparison, it is found that 215th nucleotide in the cDNA sequence of SpALF6 and 222th nucleotide G of a variant of the SpALF6, namely SpALF6-V, are nonsense mutation for each other. In the invention, the mutation site is used as the basis, a convenient and practical molecular marker detection kit is built, and a matched RT-PCR detection method is provided; detection results can be used as important indicators for judging the disease resistance of the blue crabs, and the single nucleotide polymorphism cSNP molecular marker has the great importance in predicting of the disease resistance of the blue crabs and molecule auxiliary disease resistance breeding.

Description

technical field [0001] The invention belongs to the technical field of molecular-assisted disease-resistant breeding of aquatic animals, and in particular relates to a detection kit and reverse transcription of a single nucleotide polymorphism (SNP) in the cDNA sequence of Scylla pseudocaveus SpALF6 that affects the disease resistance of the crab. A PCR (RT-PCR) detection method belongs to the detection technology of disease resistance traits in the field of aquatic crustacean breeding. Background technique [0002] Scylla paramamosain is an important marine economic crab in my country and even in the world. Pseudo-cave blue crabs, also known as green crabs, belong to the phylum Arthropoda, Crustacea, Decapod, and the genus Scylla. In my country, they are mainly distributed in the coastal waters of the southeast coast. At present, they are also cultivated in the Yangtze River estuary such as Chongming Island. It belongs to my country's traditional marine fishery. Resources a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 李新苍王元房文红周俊芳赵姝
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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