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Gene cloning of goat RanBP9 and positioning detection method of goat RanBP9 in sperm

A ranbp9 and goat technology, applied in the biological field, can solve problems such as positioning and detection blanks

Inactive Publication Date: 2016-03-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] But goat RanBP9 gene sequence and its location detection in sperm are still blank

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  • Gene cloning of goat RanBP9 and positioning detection method of goat RanBP9 in sperm
  • Gene cloning of goat RanBP9 and positioning detection method of goat RanBP9 in sperm
  • Gene cloning of goat RanBP9 and positioning detection method of goat RanBP9 in sperm

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specific Embodiment approach 1

[0016] Specific embodiment one: present embodiment mainly is the RanBP9 gene sequence of cloning goat, is characterized in that carrying out according to the following steps:

[0017] 1. Extraction of total RNA from goat testis tissue (according to the instructions of the animal tissue total RNA extraction kit) and detection of RNA integrity and purity (such as figure 1 );

[0018] 2. The cDNA template was obtained by reverse transcription, and the reaction system and procedures were carried out in the following table;

[0019] Table 1 Reverse transcription system

[0020] composition Volume (μl) 5×iScript reaction mix 4 iScript reverse transcriptase 1 Nuclease-free water 14 RNA template 1 Total volume 20

[0021] Table 2 Reverse transcription program

[0022] temperature time 25℃ 5min 42℃ 30min 85℃ 5min

[0023] 3. RT-PCR amplification of RanBP9 gene;

[0024] 1. Design primers to amplify the...

specific Embodiment approach 2

[0033] Specific embodiment two: this embodiment is the same as steps one and two of specific embodiment one, and the difference is carried out according to the following steps:

[0034] 1. Download goat β-actin gene mRNA sequence and cloned RanBP9 gene sequence on NCBI to design fluorescent quantitative PCR primers;

[0035] Table 6 goat RanBP9 gene and β-actin fluorescent quantitative PCR primers

[0036]

[0037] 2. Fluorescence quantitative detection of the relative expression of RanBP9mRNA, the specific procedure is as follows:

[0038] Table 7 Fluorescent quantitative PCR reaction system

[0039] components Volume (μl) Nuclease-Free Water 7.2 Forward Primer 0.4 Reverse Primer 0.4 GoTaq-QPCR Master Mix, 2× 10 Template cDNA 2 Total 20

[0040] Table 8 Fluorescence quantitative PCR reaction program

[0041] temperature time 95℃ 10min 95℃ 15sec 60℃ 1min 40cycles

[0042] 3. The Ct v...

specific Embodiment approach 3

[0043] Specific embodiment three: this embodiment is to carry out the tissue expression analysis of RanBP9 protein, and specific implementation steps are as follows:

[0044] 1. Sample collection: quickly collect goat testes and epididymis. Goat testicles were cut in half and fixed for a week, then trimmed into small pieces, fixed for 72 hours, trimmed again, and fixed for another 48 hours. The goat epididymis were fixed for one week according to the head of the epididymis, the body of the epididymis and the tail of the epididymis, and were trimmed into small pieces. After 72 hours of fixation, the pieces were trimmed again, and the fixation was continued for 48 hours;

[0045] 2. After the testis and epididymis samples are fixed, the neatly clothed samples are trimmed, classified and labeled, and dehydrated, transparent, waxed and embedded according to the following procedures;

[0046] Table 9 Tissue sectioning procedure

[0047] step Processing reagent Proce...

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Abstract

The invention discloses gene cloning of goat Ran binding protein 9 (RanBP9) and a positioning detection method of the goat RanBP9 in sperm and relates to the technical field of biology. The method includes the steps of firstly, designing cloning primers; secondly, performing RT-PCR amplification on a sequence, and sequencing to obtain a goat RanBP9 gene sequence; thirdly, analyzing the relative expression quantity of RanBP9 mRNA; fourthly, expressing the RanBP9 in tissue; fifthly, using a sperm cell immunofluorescence method to detect the positioning of the RanBP9 in the sperm. The method has the advantages that the goat RanBP9 gene sequence is obtained, expression of the RanBP9 RNA and the RanBP9 in different tissue and positioning of the RanBP9 in the sperm are researched, and a foundation is laid for researching the functions of the RanBP9 in the growth of goat.

Description

technical field [0001] The invention discloses goat Ran-binding protein 9 (RanBP9) gene cloning and its location detection method in sperm, and relates to the field of biotechnology. Background technique [0002] RanBP9 can develop on its own in embryonic stem cells, and has an important regulatory role in the process of sperm and oogenesis. It is also a scaffold protein composed of multiple parts, and plays a role in mediating cell surface receptor proteins and intracellular signal transmission. important function. The function of RanBP9 protein is related to various biological processes, such as cell proliferation and apoptosis, growth and development of gonads, etc. In addition, RanBP9 is essential for mouse spermatogenesis and oogenesis, and there is a significant decline of RanBP9 in the development of testis mutants during the sperm cell reproduction stage. It is also a novel binding protein of neuroreceptor, which acts as an interactor in the cytoplasm to induce sig...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C12Q1/68G01N33/68G01N33/569
CPCC07K14/47C12Q1/6851G01N33/56966G01N33/68C12Q2531/113C12Q2563/107
Inventor 赵永聚沈彦花唐毅平
Owner SOUTHWEST UNIVERSITY
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