Macrobracium rosenbergii spermatozoon gelatinases, encoding gene and application thereof

A technology of Macrobrachium rosenbergii and coding genes, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., and can solve the problems that there are no detailed reports on proteins or genes

Inactive Publication Date: 2009-02-18
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in shrimps belonging to arthropods, there are no

Method used

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  • Macrobracium rosenbergii spermatozoon gelatinases, encoding gene and application thereof
  • Macrobracium rosenbergii spermatozoon gelatinases, encoding gene and application thereof
  • Macrobracium rosenbergii spermatozoon gelatinases, encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Acquisition of the Gelatinase Encoding Gene of Macrobrachium rosenbergii Sperm

[0020] ①Use Trizol to extract total RNA from the male reproductive tract of Macrobrachium rosenbergii according to the method provided by the manufacturer. Using this as a template, the first-strand cDNA was synthesized using reverse transcriptase.

[0021] ② According to the sequence of the 40 amino acids at the amino terminal, design three degenerate primers (F1:

[0022] 5'-GAYGARAYYTNGCNWSNGARG-3', F2:

[0023] 5'-GAYYTNGCNWSNGARGTNCARCA-3' and R1:

[0024] 5'-ATYTCRTCRTCNCCNCCNGCNA-3').

[0025] ③ In the first round of PCR reaction, the first-strand cDNA was used as a template, and F1 and R1 were used as primers. The PCR reaction system is: 1 μl of cDNA template, 1 μl of each primer, 2.5 μl of 10×PCR Buffer, MgCl 2 1.5 μl, dNTPs 1.0 μl, TaqE 0.25 μl, and sterilized water to make up to 25 μl. The PCR program was denaturation at 94°C for 5 min, followed by 30 cycles at ...

Embodiment 2

[0050] Embodiment 2: Hydrolysis of Macrobrachium rosenbergii sperm gelatinase

[0051] Sample preparation to be tested:

[0052] Sample 1 (crude extract of Macrobrachium rosenbergii sperm): take the ampulla of mature male Macrobrachium rosenbergii, crush it, and centrifuge to obtain sperm, and use ultrasonic treatment for 5 minutes at 4°C, and the supernatant obtained after centrifugation is Crude sperm extracts were stored at -80°C for future use.

[0053] Sample 2 (the product of the reaction between the macrobrachium rosenbergii sperm crude extract and MRPINK): the sperm crude extract (10 μg) was reacted with MRPINK (5 mM) for half an hour at 28 ° C, and the reaction product was passed through SDS-containing 0.1% gelatin. Activity was detected by PAGE.

[0054] Sample 3 (SGP): Combined with the gelatin activity detection experiment, the SGP was separated and purified from the crude sperm extract by using the HPLC method. The elution condition of HPLC is acetonitrile cont...

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Abstract

The invention relates to protease-(M.rosenbergii sperm gelatinase-like protease, SGP) with the hydrolyzed gelatin function, which is purified from the sperm crude extract of giant freshwater prawn (M.rosenbergii), and a coded gene and an application thereof. The studies of enzyme related with fertilization on sperms of different species are helpful to understanding the mechanism of crustacean fertilization and the mechanism of the production of reproductive isolation between different species. Based on the theories and the techniques, the relevant researchers can establish a good hybrid system, thus obtaining high yield and fast growth hybrid species.

Description

(1) Technical field [0001] The present invention relates to macrobrachium rosenbergii sperm gelatinase-like protease (sperm gelatinase-like protease, SGP) and its coding gene and application. (2) Background technology [0002] During fertilization, the combination of sperm and egg has the characteristics of specificity among species. As one of the regulators of plasma membrane fusion, proteins on sperm play an important role in this process. In mammals, such as mice, a sperm protein called Izumo is a key factor in the fusion of sperm and egg plasma membranes. Another sperm surface protein, cyritestin, plays an important role in the specific adhesion of sperm to eggs. Proteins (proteases) on sperm also play an important role in the fertilization process in invertebrates. Like abalone, mussels and several snails, the sperm protein lysin is considered to be the main factor for sperm to penetrate the egg membrane. However, in shrimps belonging to arthropods, there are no det...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57
Inventor 李晔马文明杨卫军
Owner ZHEJIANG UNIV
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