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Method for recombinant production of procambarus clarkii ATP synthase F0 subunit 6 protein

A technology of ATP synthase and Procambarus clarkii, applied in the field of genetic engineering, can solve the problems of decreased egg quality, shortened molt cycle of parent body, increased mortality, etc.

Inactive Publication Date: 2016-03-02
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, there will be unavoidable side effects in practice: after removal of eye stalks, it is easy to shorten the molting cycle of the parent body, increase the mortality rate, and reduce the quality of eggs and other adverse consequences

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The acquisition of embodiment 1 Procambarus clarkii atp6 gene

[0016] The chemical synthesis of the whole gene was carried out according to the sequence of the atp6 gene of Procambarus clarkii (Sequence ID: AFQ31581) published in the GenBank database.

Embodiment 2

[0017] Example 2 Escherichia coli recombinant expression, purification and immunoblotting identification of Procambarus clarkii ATP synthase F0 subunit 6

[0018] Design primer atp6-1: (5′-TCAGGA GGTACC ATGATAACAAGATTATTTAGA-3′) and atp6-2: (5′-ACTGAG AAGCTT TTAATTTACTTCTCTAGCATATAAAG-3'), the cDNA coding frame 5' and 3' sides of the ATP synthase F0 subunit 6 gene of Procambarus clarkii were introduced into KpnI and HindIII restriction enzyme sites (shown underlined) respectively by PCR. The atp6 gene was inserted into the commercial expression plasmid pColdII by restriction enzyme digestion, molecular ligation and other genetic operations to construct the recombinant plasmid pColdII-atp6. Then pColdII-atp6 was transformed into Escherichia coli expression strain BL21(DE3), positive transformants were screened by ampicillin antibiotic plate and verified by sequencing, and then the recombinant NADH dehydrogenase subunit III protein was induced and expressed by IPTG. The mediu...

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Abstract

The invention relates to a method for recombinant production of procambarus clarkii ATP synthase F0 subunit 6 protein, and belongs to the technical field of gene engineering. According to the method, an atp6 gene is obtained through whole-gene synthesis, a recombinant expression vector pColdII-atp6 is constructed and transforms an escherichia coli expression strain BL21(DE3) to achieve efficient expression, and then an important foundation is laid for the following intensive study for the molecular mechanism of the procambarus clarkii ovary 'eyestalk excision effect'.

Description

technical field [0001] The invention relates to a genetic engineering bacterium highly expressing Procambarus clarkii ATP synthase F0 subunit 6, which belongs to the technical field of genetic engineering. Background technique [0002] "Eyestalk resection induces rapid development and maturation of shrimp and crab crustacean ovaries" (referred to as the "eyestalk resection effect" of shrimp and crab ovaries) has a similar molecular mechanism, and elucidating the molecular mechanism has important scientific significance and application prospects. How to realize the regulation of artificial ovary development with no / weak side effects has always been one of the important problems in economical shrimp and crab farming. At present, although people have explored many methods to promote the rapid maturation of shrimp and crab ovaries, such as temperature control, light control, strengthening nutritional conditions, hormone treatment, etc., eye stalk removal is still the most import...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/14C12R1/19
Inventor 水燕周鑫徐增洪沈怀舜陈丽薇
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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