Method for obtaining FSHR full-length coding region sequence with multiple splice forms

A coding region and sequence technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of heavy workload, high test cost, high cost, etc., and achieve the effect of roughly clear length, highlighting technological progress, and low cost

Inactive Publication Date: 2014-04-23
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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Problems solved by technology

1. The gene fragment length of the target PCR product is not clear
2. High test cost
3. Heavy workload and high requirements for experimen

Method used

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  • Method for obtaining FSHR full-length coding region sequence with multiple splice forms
  • Method for obtaining FSHR full-length coding region sequence with multiple splice forms
  • Method for obtaining FSHR full-length coding region sequence with multiple splice forms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0033] Step 1 extracts the total RNA (ribonucleic acid) in the sheep granulosa cells, and then reverse-transcribes the total RNA into cDNA;

[0034] 1. Collection of Tissue

[0035] Collect sheep ovaries from the slaughterhouse, keep them warm in sterilized normal saline at 30°C, and transport them to the laboratory within 2 hours. Extract granulosa cells and follicular fluid with a 10mL sterile syringe in the laboratory, and transfer the extracted follicular fluid and granulosa cells to a 1.5mL centrifuge. Centrifuge in the tube at 4°C, 4000rpm, 5min, discard the supernatant and save the precipitate, which is used for RNA extraction.

[0036] 2. RNA preparation and cDNA synthesis

[0037] Add 1.0mL Trizol solution to the 1.5mL EP tube precipitation, pump repeatedly 10-20 times with a 1mL sterilized syringe until the cell lysis solution is completely clear, and place at room temperature for 5min.

[0038] Extract sheep follicle fluid with a 10mL syringe needle, sort out oocy...

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Abstract

The invention provides a method for obtaining an FSHR (follicle-stimulating hormone receptor) full-length coding region sequence with multiple splice forms. The method comprises the steps: extracting total RNA of a sheep granular cell, and reversely transcribing into cDNA; according to initiation codon upstream and termination codon downstream of a cDNA sequence of a sheep FSHR gene, designing two pairs of primers, wherein an upstream primer F1 is CAAAAGGGCTCAGTGTGGAG, an upstream primer F2 is CGTCTGCAGAAGCAGAAGCA, a downstream primer R1 is CTTATGGATGTGCCAGGGAG, and a downstream primer R2 is AGTGCTCTGTCAGCTCTTGC; taking the obtained cDNA as a template, carrying out PCR amplification of the FSHR gene, and extracting the PCR product by a Tiangen gel extraction kit; and adding an A tail into the PCR product, and cloning, sequencing and verifying an T vector. The obtained FSHR full-length coding region sequence with the multiple splice forms is characterized by comprising the oFSHR695, oFSHR694, oFSHR648, oFSHR633 and oFSHR595.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for obtaining the complete sequence of multiple spliced ​​forms of the sheep follicle-stimulating hormone receptor (FSHR) gene. Background technique [0002] Studies have found that follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor mainly present on the membranes of vertebrate ovary granulosa cells and testis Sertoli cells, and plays a role in mediating FSH regulation of individual pubertal sexual maturation. , Stimulating the physiological process of germ cell maturation plays an important role, in addition, FSHR also plays an important role in osteoclasts (Cell.2006Apr21;125(2):247-260.), monocytes (Biochem Biophys Res Commun .2010Mar26;394(1):12-17.), oocyte (J Clin Endocrinol Metab.2002May;87(5):2266-2276.), tumor vessel wall (N Engl J Med.2010Oct21;363(17 ):1621-1630.) are also expressed, but the biological functions expressed at these positi...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 吴阳升黄俊成蒋香菊林嘉鹏汪立芹刘明军陆立里
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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