Method for utilizing buffalo lactation related gene Leptin as molecular marker and application of method

A technology of molecular markers and genotyping, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as the absence of buffalo Leptin gene, and achieve the effect of improving production performance

Active Publication Date: 2016-05-04
GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, so far, there is no research report on buffalo Leptin gene as a molecular marker of buffalo milk production traits

Method used

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  • Method for utilizing buffalo lactation related gene Leptin as molecular marker and application of method
  • Method for utilizing buffalo lactation related gene Leptin as molecular marker and application of method
  • Method for utilizing buffalo lactation related gene Leptin as molecular marker and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Preparation of buffalo genome total DNA

[0028] 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:

[0029] 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 inversion, centrifuge at 12,000 rpm for 1 min, absorb the supernatant, and leave the precipitate; add an equal volume of cell lysate CL, repeat this step Once; add 4μL RNAase (100mg / mL), shake for 15sec, and let stand at room temperature for 5min;

[0030] 2. Add 20 μL Proteinase K solution, mix well, add 200 μL buffer GB, fully invert and mix, place at 56°C for 10 minutes, invert and mix several times during the period, the solution should become clear;

[0031] 3. Add 200 μL of absolute ethanol and mix thoroughly by inversion; flocculent precipitation may appear at this time;

[0032] 4. Add the solution and flocculent preci...

Embodiment 2

[0038] Example 2: Acquisition of Leptin Gene Molecular Marker for Buffalo Milk Production Traits

[0039] 1. Primer design: using SEQNO: 1 as a template, using Primer5.0 primer design software, detected by Oligo software, determined the forward direction primer for amplifying the buffalo Leptin 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: 2-3.

[0040] 2. PCR amplification reaction

[0041] Genomic DNA samples from 50 buffaloes were randomly selected for pooling as templates for PCR reactions.

[0042] (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 17 μL of ddH2O.

[0043] (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

[0044] 3. PCR product sequencing, analysis and mol...

Embodiment 3

[0058] Example 3: Application of buffalo milk production trait gene Leptin as a molecular marker

[0059] The species of buffaloes in the test group are individual hybrid milk buffaloes in my country. The association analysis between different genotypes of Leptin gene G3683T locus and milk production traits was carried out in buffalo herd.

[0060] The results of association analysis of buffalo LeptinG3683T loci are shown in Table 2. It can be concluded from the table that the genotype is significantly correlated with the milk production traits of buffalo (P<0.05), and the milk production of buffalo individuals carrying GT genotype is higher than that of GG and TT individuals, which is the dominant genotype.

[0061] Table 2 Association analysis of Leptin gene G3683T site and buffalo milk production

[0062]

[0063] Note: For the same data, different letters indicate extremely significant difference (P0.05).

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Abstract

The invention belongs to the technical field of preparation of livestock molecular markers, and particularly relates to an application of a buffalo milk production trait related Leptin gene G3683T locus as a molecular marker to buffalo lactation performance research. The method is characterized in that a nucleotide sequence of a Leptin gene fragment is represented as SEQ ID NO:1, wherein the 3683th locus has G/T base mutation, the locus is remarkably related with milk production of buffalo species, and P is smaller than 0.05. The invention discloses sequences of upstream and downstream primers required for detecting the G/T base mutation at the 3683th locus represented as a sequence table SEQ ID NO:1. The novel molecular marker method is provided for buffalo milk production trait markers.

Description

【Technical field】 [0001] The invention belongs to the technical field of livestock molecular marker preparation, and in particular relates to a method for using buffalo milk production trait gene Leptin as a molecular marker and its application. 【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,...

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 庞春英邓廷贤梁贤威陆杏蓉朱鹏段安琴
Owner GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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