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Trypsin gene cDNA sequence of Scylla paramamosain and cloning method and application thereof

A technology of trypsin gene and mud crab, which is applied in the field of cDNA sequence and cloning of the trypsin gene of mud crab, can solve the problem of estimating the amount of feeding, optimize the seedling breeding process, improve the survival rate of seedlings, The effect of reducing bait consumption

Inactive Publication Date: 2010-09-22
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there have been no reports on estimating the amount of feeding and predicting the molting cycle of blue crabs based on the expression level of trypsin gene in blue crabs

Method used

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  • Trypsin gene cDNA sequence of Scylla paramamosain and cloning method and application thereof
  • Trypsin gene cDNA sequence of Scylla paramamosain and cloning method and application thereof
  • Trypsin gene cDNA sequence of Scylla paramamosain and cloning method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of trypsin gene from Scylla syringae

[0037] (1) RNA isolation (RNA isolation)

[0038] Take the hepatopancreas of Scylla pseudocavena, add liquid nitrogen, grind it with a mortar, add it to a 1.5mL EP tube filled with Trizol lysate, oscillate fully, extract total RNA (TRIzol Reagents, TaKaRa), and use formaldehyde deformable gel electrophoresis Identify the quality of total RNA, and then measure the RNA content on a spectrophotometer;

[0039] Proceed as follows:

[0040] 1. Homogenization

[0041] Add 10-30mg of tissue to 1ml TRIzol, and use an electric homogenizer or disposable grinding pestle to fully homogenize;

[0042] 2. Phase Separation

[0043] a. After homogenization, place at room temperature for 5 minutes to fully lyse the sample, then centrifuge at 12,000 rpm for 10 minutes at 4°C, and take the supernatant;

[0044] b. Then add 200 μl of chloroform, vibrate vigorously and mix well, then place at room temperature for 3-5min to allow natural ph...

Embodiment 2

[0069] Fluorescence Quantitative PCR Detection of Trypsin Gene in Scylla sclerophyllum

[0070] Application in Predicting Feed Amount and Moulting Cycle of Scylla Seedlings

[0071] (1) Total RNA extraction

[0072] Take individual blue crabs at each daphnia stage, add liquid nitrogen, grind with a mortar, add 1.5mL EP tube containing lysate, shake fully, extract total RNA (TRIzol Reagents, TaKaRa), use formaldehyde deformable gel Identify the quality of total RNA by electrophoresis, and then measure the RNA content on a spectrophotometer;

[0073] (2) Fluorescent quantitative PCR experiment

[0074] According to TaKaRa's SYBR According to the instructions of ExScript TM RT-PCR Kit (Perfect Real Time), the reverse transcription conditions are 42°C for 15 minutes and 95°C for 2 minutes;

[0075] Take the reverse-transcribed cDNA extracted from samples of each daphne larvae stage of Scylla syringae, and take 2 μl and dilute it with EASY Dilution (TaKaRa Code: D9160, Shiga, ...

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Abstract

The invention relates to a trypsin gene cDNA sequence of Scylla paramamosain and a cloning method and application thereof. The trypsin gene cDNA sequence of Scylla paramamosain has the sequence shown in SEQ ID NO:1; the cloning thereof comprises the following steps: extracting total RNA from the hepatopancreas of the Scylla paramamosain; building a cDNA plasmid library; carrying out authentication and sequencing analysis on the cDNA plasmid library; and according to the BLAST comparison, obtaining the sequence shown in SEQ ID NO:1. The trypsin gene cDNA sequence of the Scylla paramamosain can forecast feeding amount and exuviation cycle in the germchit and reproductive process of blue crabs so that people can master the life cycle of the blue crabs and select and use in inheritance breeding, reduce the bait consumption, improve the germchit survival rate, thereby optimizing the process of grow seedlings.

Description

technical field [0001] The invention belongs to the field of gene coding, cloning and application of mud crab, and in particular relates to a cDNA sequence of trypsin gene of mud crab and its cloning method and application. Background technique [0002] Crustacean growth and development are always associated with moulting. The whole process of molting includes shedding the old carapace, the individual grows rapidly due to water absorption, and then the new carapace is formed and hardened. Therefore, the individual growth of crustaceans is discontinuous in shape, showing a ladder shape, and every time a shell is shed, a step is raised. The molting cycle can be divided into molting, post-molting, inter-molting and pre-molting. For crustaceans, according to the standards of Drach and Tchemigovtzef, the molt cycle can be further divided into E, A, B, C (C 1 -C 4 ), D (D 0 -D 4 ) 5 phases. The growth of blue crab is also discontinuous, and moulting is a sign of its growth....

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/76C12N15/10C12Q1/68
Inventor 马凌波蒋科技张丹张凤英乔振国皮妍马春艳石彦红陶启长郑俊斌
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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