SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof

A technology of heat resistance and site, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problem of SNP screening related to heat resistance, and achieve the effect of shortening the breeding time, low cost and strong purpose

Inactive Publication Date: 2015-02-04
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research on heat tolerance of flounder and flounder, the main focus is on the screening of microsatel

Method used

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  • SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof
  • SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof
  • SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Flounder HSP70 gene cDNA and DNA sequence cloning in the present invention comprise the following steps:

[0017] a) extraction of total RNA from the heart of flounder and extraction of muscle DNA;

[0018] b) synthesis of cDNA first strand;

[0019] c) Obtaining the full-length cDNA sequence and DNA sequence of the target gene.

[0020] The specific operation is as follows:

[0021] a) Extraction of total RNA from flounder. Total RNA was extracted from flounder heart according to the method of Trizol (Invitrogen, CA, USA); muscle DNA was extracted by phenol / chloroform extraction.

[0022] b) Synthesis of cDNA first strand. Using 2 μg of total RNA as a template, add 1 μl of 20 μMOligo d(T) respectively, make up to 13 μl with DEPC water, mix well and spin off. Incubate at 70°C for 10 min, immediately place on ice, add in sequence: 5 μl 5×M-MLV Buffer, 5 μl IdNTP (2.5 mM), 1 μl RNasin (40U / μl), 1 μl M-MLV (200U / μl); after mixing and centrifuging, 42°C, 90min. Store...

Embodiment 2

[0027] Example 2 Screening of SNP sites related to heat resistance in flounder HSP70 gene

[0028] The screening and correlation analysis of SNP loci related to heat tolerance traits in the HSP70 gene of flounder flounder include the following steps:

[0029] a) Construction of flounder family

[0030] b) Temperature tolerance experiment of flounder

[0031] c) Acquisition of relatively heat-resistant and heat-labile trait populations

[0032] d) Screening and correlation analysis of SNP sites

[0033] The specific operation steps are as follows:

[0034] a) Construction of flounder family

[0035] The first-generation population with good growth traits selected in the family was taken as the parents, and individuals without genetic relationship were selected for random combination. A half-sib family was established with a male-to-male ratio of 2:1; a full-sib family was established with a male-to-male ratio of 1:1. All families were cultured under the same culture condi...

Embodiment 3

[0047] Example 3, the application of SNPs in screening heat-resistant individuals / family

[0048] 1. Screening of heat-resistant broodstock

[0049] The fin rays of the parents to be screened were cut, DNA was extracted, the parental HSP70 gene sequence was amplified with primers HSP70fw1 and HSP70rv1, and sequenced, the SNP site genotype of the HSP70 gene was detected, and the parent fish that matched the genotype as heat-resistant were selected.

[0050] 2. The construction of the family line

[0051] The whole-sib and half-sib families were constructed with broodstock whose genotype was heat-resistant, and the family with unscreened broodstock was used as the control group.

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Abstract

The invention relates to an SNP (single-nucleotide polymorphism) locus related to heat resistance of Paralichthys olivaceus. The SNP locus is 47th/48th site (the base is AA or A/DEL), 65th site (the base is T or C), 185th site (the base is C or T), 260th site (the base is C or T), 305th site (the base is T or A), 600th site (the base is T or C) or 815th site (the base is C or T) of HSP70 gene with the nucleic acid sequence of SEQ ID NO:1. The SNP locus is used for screening the heat-resistant individual/family of Paralichthys olivaceus. The heat-resistant family is screened by a molecular marker process by using the heat shock protein gene HSP70; the hereditary characters of all the families can be evaluated on the molecular level, and the mutant site of the genome can be clearly identified; and the relevance between the mutant site and heat resistance is analyzed by combination of the heating experiment to perform reasonable judgment on the heat-resistant molecular mechanism. Compared with the traditional breeding method, the method provided by the invention has the advantages of higher purposiveness and lower cost, and can obviously shorten the breeding time.

Description

technical field [0001] The invention belongs to the technical field of screening heat-resistant families of fish in aquaculture, and in particular relates to a SNP site related to the heat resistance of flounder and its application. Background technique [0002] Flounder (Paralichthysolivaceus), commonly known as tooth piece and partial mouth, is a cold-temperate benthic fish and an important seawater economic fish in my country. The optimum temperature for the cultivation of flounder larvae is 17-20°C, the optimum temperature for the growth of adult fish is 14-23°C, and the optimum temperature is 21°C. Flounder can survive for a short time at a water temperature as low as 1°C and as high as 33°C. Compared with larvae, the high temperature resistance of adult fish will be significantly reduced, the food intake will be reduced above 23°C, the growth will stop above 25°C, and a large number of deaths will be caused by long-term exposure to 27°C. Temperature has a significant...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张全启刘金相于海洋王旭波刘旭东齐洁王志刚贺艳
Owner OCEAN UNIV OF CHINA
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