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Buffalo lactation related INSIG2 (insulin induced gene 2) and application of buffalo lactation related INSIG2 serving as molecular marker

A technology of molecular markers and genotyping, which is applied in the direction of recombinant DNA technology, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem that there is no Buffalo INSIG2 gene, etc., and achieve the effect of improving production performance

Active Publication Date: 2016-04-27
GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, so far, there is no research report on the buffalo INSIG2 gene as a molecular marker for buffalo milk production traits

Method used

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  • Buffalo lactation related INSIG2 (insulin induced gene 2) and application of buffalo lactation related INSIG2 serving as molecular marker
  • Buffalo lactation related INSIG2 (insulin induced gene 2) and application of buffalo lactation related INSIG2 serving as molecular marker
  • Buffalo lactation related INSIG2 (insulin induced gene 2) and application of buffalo lactation related INSIG2 serving as molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Preparation of buffalo genome total DNA

[0033] The blood buffalo genome total DNA (Tiangen, Beijing) was prepared using a blood genome DNA extraction kit, and the specific operation steps were as follows:

[0034] (1) Take 500 μL of whole blood and place it in a 1.5 mL centrifuge tube, add an equal volume of cell lysate CL, mix by inverting, centrifuge at 12000 rpm for 1 min, absorb the supernatant and leave the precipitate; add an equal volume of cell lysate CL, repeat the process Step once; add 4μL RNAase (100mg / mL), shake for 15sec, and let stand at room temperature for 5min;

[0035] (2) Add 20 μL of Proteinase K solution, mix well, add 200 μL of buffer GB, fully invert and mix, and place at 56°C for 10 minutes, during which time invert and mix several times, the solution should become clear;

[0036] (3) Add 200 μL of absolute ethanol and mix thoroughly by inversion; flocculent precipitation may appear at this time;

[0037] (4) Add the solution o...

Embodiment 2

[0043] Example 2: Acquisition of INSIG2 Gene Molecular Marker for Buffalo Milk Production Traits

[0044] 1. Primer design: using SEQNO: 1 as a template, using Primer5.0 primer design software, detected by Oligo software, determined the forward primer for amplifying the buffalo INSIG2 gene, and entrusted Dalian Bao Biotechnology Co., Ltd. to synthesize it. The DNA sequences of the forward and reverse primers used to amplify the gene are shown in SEQ ID NO: 2-5.

[0045] 2. PCR amplification reaction

[0046] Genomic DNA samples from 50 buffaloes were randomly selected for pooling as templates for PCR reactions.

[0047] (1) PCR reaction: The total reaction volume is 40 μL, including 1 μL of buffalo DNA pool template, 20 μL of 10XLATaqMix, 2 μL of primer mixture (10 mMeach) and ddH 2 O17 μL.

[0048] (2) PCR reaction program: 94°C / 3min; 94°C / 30sec, 60°C / 30sec, 72°C / 1.5min, 30 cycles; 72°C for 10min; 4°C storage

[0049] 3. PCR product sequencing, analysis and molecular marker...

Embodiment 3

[0066] Example 3: Gene INSIG2 related to buffalo lactation and its application as a molecular marker

[0067] The breeds of the test buffalo herd are Moura buffalo, Niri-Rafi buffalo and their hybrid buffalo herd. The association analysis between different genotypes of INSIG2 gene A1258G, A1350C, G12529A, C12670T loci and milk production traits was carried out in buffalo herd.

[0068] Table 2 shows the results of association analysis of buffalo INSIG2A1258G, A1350C, G12529A, and C12670T loci. From Table 2, it can be concluded that the genotype is significantly correlated with the milk production traits of buffalo (P<0.05). For the A1258G locus, the milk yield of Mora buffalo individuals carrying AA genotype was higher than that of GA and GG type individuals; the milk yield of hybrid buffalo individuals carrying GG genotype was higher than that of GA and AA individuals. For the A1350C locus, the milk yield of Mora buffalo individuals carrying AA genotype was higher than that...

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Abstract

The invention provides a buffalo lactation related INSIG2 (insulin induced gene 2) and application of the buffalo lactation related INSIG2 serving as a molecular marker, and belongs to the field of molecular markers for livestock. A nucleotide sequence of a gene segment is shown as SEQ ID NO:1, wherein A1258G, A1350C, G12529A and C12670T at 1258bpth, 1350bpth, 12529bpth and 12670bpth positions are in single base mutation, and the sites are highly related to milk yield of Murrah buffalo and hybrid buffalo. Upstream and downstream primer sequences needed for testing all the base mutation sites of the sequence table SEQ ID NO:1 are disclosed. By application of the buffalo lactation related INSIG2, milking performance of the buffalo can be increased, and technical support is provided and theoretical foundation is laid for breeding milk buffalo species with high milk yield.

Description

【Technical field】 [0001] The invention relates to the technical field, in particular to the field of animal molecular markers. It specifically relates to the gene INSIG2 related to buffalo lactation and its application as a molecular marker. 【Background technique】 [0002] Buffalo is a unique breeding stock resource in southern my country, but its low milk yield and reproductive performance have become two major scientific problems affecting its industrial development. Obviously, the use of advanced molecular marker technology in the selection of high-quality milk buffalo germplasm resources will effectively improve the efficiency of selection. [0003] Milk production trait is an important economic trait of milk buffalo, which is determined by a variety of microgenes and is a typical quantitative trait controlled by polygenes. Clone milk buffalo lactation performance-related genes and explore their genetic characteristics to improve milk buffalo production performance, pr...

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 邓廷贤梁贤威庞春英朱鹏陆杏蓉段安琴
Owner GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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