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Cloning and application of CDC42 gene molecular marker related to porcine reproductive traits

A molecular marker, pig reproduction technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc.

Active Publication Date: 2018-09-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the SNP is located at the miRNA binding site in the cis-element, different genotypes of the SNP site will affect the binding efficiency of the miRNA and the target gene, thereby affecting the regulation of the miRNA on the target gene

Method used

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  • Cloning and application of CDC42 gene molecular marker related to porcine reproductive traits
  • Cloning and application of CDC42 gene molecular marker related to porcine reproductive traits
  • Cloning and application of CDC42 gene molecular marker related to porcine reproductive traits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of CDC42 gene

[0032] (1) Primer design

[0033] Use the 3'UTR sequence of the porcine CDC42 gene provided in NCBI as the reference sequence to design the following primers for PCR amplification, and the nucleotide sequence of the primer pair is as follows:

[0034] Primer combination sequence for amplifying the CDC42 gene fragment:

[0035] Forward primer: 5′-TGCCAAGAATAAACAGAAGCCTA-3′

[0036] Reverse primer: 5′-TTGTGAAGGGTTTTTTGTTTGTTTAATAC-3′

[0037] (2) Cloning and sequencing of PCR products

[0038] The purified PCR product was connected to the pMD-18T vector (purchased from Treasure Bioengineering Dalian Co., Ltd.) overnight at 4°C in a water bath; under sterile conditions, 100-120 μl of competent cells were placed in a 1.5ml Ependorff tube, and 5 μl of The ligation product was added and mixed, placed on ice for 30 minutes, heat-shocked at 42°C for 90s, ice-bathed for 3-4 minutes, added 400 μl LB liquid medium without antibiotics, and incu...

Embodiment 2

[0039] Example 2 Immunohistochemical test of CDC42 gene fragment in placental tissue

[0040] (1) Collection of pig placental tissue samples

[0041] Pregnant sows (Large White pig breed, from the boutique pig farm of Huazhong Agricultural University) were selected and slaughtered to collect pig placental tissue samples on the 26th, 50th, and 95th days after pregnancy.

[0042] (2) Preparation of paraffin tissue sections

[0043] Take the tissue and fix it in 10% neutral formalin fixative for about 48 hours, then trim the tissue block, put the tissue into the tissue frame quickly, rinse it with tap water overnight, and then soak it in distilled water for 10 minutes. Soak the tissue in low-concentration alcohol to high-concentration alcohol in sequence, namely 70%, 80%, 90%, 95% (I, II), absolute ethanol (I, II) for 2 hours each. Put the tissue into xylene (Ⅰ, Ⅱ) until transparent, the time is about 15min each. The transparent tissues were placed in paraffin wax (Ⅰ, Ⅱ) at 60...

Embodiment 3

[0048] Embodiment 3 Establishment of PCR-RFLP diagnostic method

[0049] (1) Design the primer combination shown below

[0050] Forward primer: 5'-TGGGTGGAACCTGTCTTGCC-3',

[0051] Reverse primer: 5'-AAGCATTGGTTCAACACTAA-3'.

[0052] (2) PCR amplification conditions

[0053] The total volume of the PCR reaction is 10 μl, including about 100 ng of porcine genomic DNA, 5 μl of PCR Mix, 0.2 μl each of forward primer and reverse primer (from this example). PCR amplification program: 94°C for 5min, 32 cycles of 94°C for 30s, 60°C for 30s, then 72°C for 15s, and finally 72°C for 5min. The PCR reaction product was detected by 2% agarose gel electrophoresis, and a 682bp specific amplification fragment (ie, SEQ ID NO: 1) was obtained. Sequencing results revealed that the 151bp fragment had a Hpy188III restriction site, which was the polymorphic site.

[0054] (3) PCR-RFLP detection conditions

[0055] The volume of PCR product digestion reaction is 10 μl, including 1 μl of 1× buf...

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Abstract

The invention belongs to the technical field of screening of porcine molecular markers, and in particular relates to cloning and application of a CDC42 gene molecular marker related to porcine reproductive traits. The molecular marker is related to porcine reproductive traits such as litter size and birth weight traits. The molecular marker is cloned from a gene CDC42 which affects the developmentof the placenta and the embryo; the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1; a C / T allele mutation exists at 151bp of the sequence and causes Hpy188III-RFLP polymorphism.The invention also discloses the application of the molecular marker in correlation analysis of the porcine reproductive traits.

Description

technical field [0001] The invention belongs to the technical field of porcine molecular marker preparation, and in particular relates to the cloning and application of a porcine reproductive trait-related CDC42 gene molecular marker. The molecular markers are associated with swine reproductive traits such as litter size and birth weight. The molecular marker is cloned from the porcine CDC42 gene, and includes the detection method and application of the 3'UTR sequence mutation site of the porcine CDC42 gene. Background technique [0002] In the pig industry, the traits of birth weight and litter size are important manifestations of the economic value of pigs. How to increase the litter size of sows and the birth weight of piglets is particularly important in actual production. Sow placenta function is an important factor affecting sow litter size and piglet birth weight. During pregnancy, pig placenta increases the exchange area between mother and fetus through the unique w...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 余梅邓大栋刘睿泽刘向东李新云赵书红李小平
Owner HUAZHONG AGRI UNIV
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