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Microsatellite sequences of aeromonas hydrophila resistance related Chinese soft-shelled turtles and application of microsatellite sequences

A technology of Aeromonas hydrophila and microsatellite sequence, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve high polymorphism and stability

Active Publication Date: 2016-02-17
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the "strand slippage" phenomenon in the process of DNA replication may cause the high polymorphic information content (PIC) of microsatellite DNA

Method used

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  • Microsatellite sequences of aeromonas hydrophila resistance related Chinese soft-shelled turtles and application of microsatellite sequences
  • Microsatellite sequences of aeromonas hydrophila resistance related Chinese soft-shelled turtles and application of microsatellite sequences
  • Microsatellite sequences of aeromonas hydrophila resistance related Chinese soft-shelled turtles and application of microsatellite sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 Establishment of the method for Aeromonas hydrophila challenge

[0018] 1. Experimental sample collection

[0019] Select 200g / only Yellow River population of Chinese soft-shelled turtle, do challenge virus treatment with Aeromonas hydrophila T4 bacterial strain, the first 50 that die are susceptible group, the last 50 that die are anti-Aeromonas hydrophila group ( Among them, the soft-shelled turtle resistant to Aeromonas hydrophila was still very active after 5-6 injections of bacteria). After slaughter, the muscle tissue was stored at -80°C.

[0020] 2. Test method

[0021] Aeromonas hydrophila (Aeromonashydrophila) T4 strain was donated by Professor Lu Chengping of Nanjing Agricultural University. After rejuvenation, the T4 strain was inoculated on ordinary nutrient agar medium, cultured at a constant temperature of 28°C for 12 hours (OD6008 cfu / mL, use 1.0×10 8 cfu / 50g body weight concentration to attack soft-shelled soft-shelled turtle.

[0022]...

Embodiment 2

[0023] The acquisition of embodiment 2 microsatellite site and primer design

[0024] 1. Extraction of DNA in the susceptible group and the anti-Aeromonas hydrophila group

[0025] Using phenol extraction and ethanol precipitation of DNA:

[0026] (1) Take 0.025g soft-shelled turtle muscle sample and put it into a 1.5mL centrifuge tube. Add 100mL of STE lysis buffer, cut it into small pieces with small ophthalmic scissors on ice, then add 400mL of STE lysis buffer, 50μL SDS (10%), 5μL proteinase K (20mg / mL), mix well, put it into 56℃ for 4h digestion, Mix once during 1h. until the lysate is clear.

[0027] (2) Add 500 mL of phenol / chloroform / isoamyl alcohol (25:24:1), extract for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes.

[0028] (3) Take the centrifuged supernatant and repeat the above steps once until there is no protein layer between the aqueous phase and the organic phase.

[0029] (4) Add an equal volume of 500 μL of chloroform / isoamyl alcohol (24:1) t...

Embodiment 3

[0043] Example 3 Carrying out PCR amplification of soft-shelled turtle microsatellite loci

[0044] The following conditions are used to amplify the DNA of the susceptible group and the anti-Aeromonas hydrophila group: see Table 2 and Table 3 for details

[0045] Table 2 PCR amplification system

[0046]

[0047] Table 3PCR amplification conditions

[0048]

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Abstract

The invention relates to microsatellite sequences of aeromonas hydrophila resistance related Chinese soft-shelled turtles and an application of microsatellite sequences. According to the microsatellite sequences, the nucleotide sequence is represented as SEQ ID NO.1, and primer sequences are represented as SEQ ID NO.2 and SEQ ID NO.3. The microsatellite sequences and primers suitable for screening aeromonas hydrophila resistant Chinese soft-shelled turtle individuals are developed, the specific primers are designed on the basis of the nucleotide sequence of a microsatellite, further, relation of the microsatellite molecular marker with the aeromonas hydrophila resistant capacity of the Chinese soft-shelled turtles is discovered, the aeromonas hydrophila resistant Chinese soft-shelled turtle individuals can be screened out through genotyping, and microsatellite loci have high polymorphism and stability and can be further applied to molecular assisted selection of the Chinese soft-shelled turtles.

Description

technical field [0001] The invention belongs to the technical field of molecular biology DNA markers, and in particular relates to a microsatellite locus of soft-shelled turtle anti-Aeromonas hydrophila and an application thereof. Background technique [0002] Soft-shelled turtle, also known as soft-shelled turtle, tortoise, water fish, yuan fish, turtle, foot fish and old soft-shelled turtle, English name Chinese Soft-shelled Turtle, Latin name (Pelodiscus sinensis), China except Ningxia, Xinjiang, Qinghai and Tibet have not been reported , and the rest of the provinces, municipalities, and autonomous regions are distributed, especially in the Yangtze River Basin and South China; abroad are distributed in Japan, Korea, and Vietnam. The Chinese soft-shelled turtle has been consumed in large quantities for a long time because of its medicinal value and nutritional value, and the variety is one of the material basis (variety, feed, water quality) of Chinese soft-shelled turtle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 王伟钱国英李彩燕尹尚军葛楚天徐建陈忠法汪财生
Owner ZHEJIANG WANLI UNIV
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