Bovine embryo sex development related gene, encoding protein and preparation method thereof

A technology for encoding proteins and genes, applied in the field of molecular biology

Inactive Publication Date: 2010-12-15
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no gender-based controls have been successfully applied in agriculture

Method used

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  • Bovine embryo sex development related gene, encoding protein and preparation method thereof
  • Bovine embryo sex development related gene, encoding protein and preparation method thereof
  • Bovine embryo sex development related gene, encoding protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Extraction of total RNA from bovine early fetal genital ridge

[0090] Use TaKaRa RNAiso Reagent (Code No.D312) to extract the total RNA of bovine early fetal genital ridge, and use DNase I (RNase Free) (Code No. D2215) for DNase I treatment, and finally dissolve in DEPC-treated water. Take 1ul for 1% agarose gel electrophoresis, see the results figure 1 , wherein: M: DL2000 DNA Marker; 1: Total RNA from bovine bovine early fetal genital ridge (DNase I treatment).

Embodiment 2

[0091] Example 2: Known sequence verification

[0092] 1. Primer Design and Synthesis

[0093] name

sequence

sequence

CTD026XF4:

5'-AATCTCCTGGACCCCTTCAT-3'

20mers

CTD026XR4:

5'-CGGCGCGGCTGGTACTTGTAG-3'

21mers

[0094] 2. Reverse transcription

[0095] cDNA was synthesized using TaKaRa 3′-Full RACE Core Set Ver.2.0 (Code No.D314), and M-MLV(-) control was set up at the same time.

[0096] reaction system:

[0097] Total RNA (1ug / ul)

1ul

Random 6mers(5uM)

1ul

dNTP Mixture (10mM each)

1ul

RNase Free dH20

4.5ul

[0098] Reaction conditions: 70°C, immediately place on ice for 2 minutes after 10 minutes

[0099] Then add the following components:

[0100]

[0101] Reaction conditions: 30°C, 10min; 42°C, 60min; 70°C, 15min

[0102] 3.PCR amplification

[0103] Use TaKaRa LA (Code No.DRR002A), carry out PCR amplification.

[0104] reaction system:

[0105] ...

Embodiment 3

[0116] Example 3: 5' end sequence acquisition

[0117] (1) 5′ RACE

[0118] 1. Primer Design and Synthesis

[0119] Primers for 5' RACE were designed based on the sequences obtained above.

[0120] name

sequence

sequence

CTD026R692:

5'-CGCTTGACGTGCGGCTTGTTC-3'

21mers

CTD026R619:

5'-CCTTGAGCACCTGGCTGACGG-3'

21mers

[0121] 2.5′RACE pretreatment and reverse transcription

[0122] Use TaKaRa 5′-Full RACE Kit (Code No.D315) to treat 3ug total RNA of bovine early fetal genital ridge with CIAP and TAP, connect with 5′RACE Adapter, reverse transcribe and synthesize cDNA, and establish M-MLV ( -) Control.

[0123] reaction system:

[0124]

[0125] Reaction conditions: 30°C, 10min 42°C, 60min 70°C, 15min

[0126] 3.PCR amplification

[0127] Use TaKaRa LA (Code No.DRR002A), carry out PCR amplification.

[0128] Outer PCR reaction system:

[0129]

[0130] Reaction conditions

[0131] 94℃ 3min 1cycle

[0132]

...

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PUM

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Abstract

The invention discloses a bovine embryo sex development related gene SOX9, relating to the biological field. A nucleotide sequence of the related gene is as shown by SEQ ID NO.1. The invention also provides an encoding protein of the gene, and an amino acid sequence of the encoding protein is as shown by SEQ ID NO.3. The invention also provides an extracting method of the gene, comprising the following steps of: extracting a total RNA (Ribonucleic Acid) from a bovine embryo genital ridge, amplifying by an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, and cloning to a complete expressed sequence of the bovine embryo sex development related gene SOX9 by combining an RACE and nest-type PCR technology. The gene can be used for controlling the sex of a bovine offspring and has good application prospect.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a gene related to sex development of early bovine fetus, encoded protein and a preparation method thereof. Background technique [0002] For hermaphrodites, sex determination and sex differentiation determine the existence of two sexes, female and male, and are an indispensable part of the normal development and survival of individuals, as well as the basis for the reproduction and continuation of races. [0003] In 1990, Sinclair et al. cloned into the human "sex determining region (SRY) on the Y chromosome". Koopman et al. transplanted a DNA fragment (14kb) containing the SRY gene into XX mice, and the mice developed into males. The experimental results revealed the fundamental way of animal sex control. However, sex determination is an intricate process. Sex determination and differentiation are an orderly and coordinated expression process dominated by the SRY gene, involvin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C07K14/47
Inventor 杜卫华朱化彬徐超
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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