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Beta-agarase encoding gene and gene acquisition method

An agarase and gene technology, applied in the field of genetic engineering, can solve the problems of limited sequences, large differences, difficult to obtain core sequences, etc., and achieve the effect of low cost

Active Publication Date: 2010-09-29
SECOND INST OF OCEANOGRAPHY MNR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large number of agarase families, the limited number of published sequences, and the large differences between them, it is difficult to obtain the core sequence according to common cloning methods

Method used

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  • Beta-agarase encoding gene and gene acquisition method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Acquisition of the core sequence of the β-agarase gene aga41

[0022] According to the different families of agarases GH16, GH50, GH86 and GH96, perform amino acid multiple sequence alignment in the software ClusterW to find the conserved sequence, and at the same time compare and find the conserved region in the software Block Maker, using the software CODEHOP Search the degenerate primers in the conserved region, and select the appropriate degenerate primers for PCR amplification. By amino acid sequence alignment in this way, the degenerate primers designed from the conserved sequences NWGFTSFGNW and ENYNVGFVSV of the GH50 family (upstream primer GH50F: 5'-AACTGGGGCTTCACCACCYTNGGNAAYTGG-3'; downstream primer GH50R: 5'-ACACGAAGCCCACGTTCTARTTYTCNCC-3') to The sodium-demanding Vibrio (preservation number: CGMCC No.2428) reported in Chinese patent 200810121997.0 was used as a template for PCR amplification, and a core sequence of β-agarase gene aga41 was obtain...

Embodiment 2

[0023] Example 2: Acquisition of the flanking sequence of the β-agarase gene aga41

[0024] According to the core sequence of β-agarase gene aga41 obtained in Example 1, the flanking sequence was amplified by site-stepping PCR technique. The specific method is as follows:

[0025] Design 3 specific nested primers based on the core sequence to amplify the 5' upstream sequence:

[0026] GSP1: 5'-ATACAAACCACTACGCACTGGCGATT-3';

[0027] GSP2: 5'-GCAATACCGTCAACAATACAAACCACTA-3';

[0028] GSP3: 5'-GATTACGGGAGACCACCAACGAGTTAGT-3';

[0029] Synthetic anchor sequence sites:

[0030] 5'-CACGACACGCTACTCAACACACCACCTCGCACAGCGTCCTCAAGCGGCCGCNNNNNNNGCCT-3';

[0031] Synthetic specific nested primers:

[0032] SFP1: 5'-CACGACACGCTACTCAACAC-3';

[0033] SFP2: 5'-ACTCAACACACCACCTCGCACAGC-3');

[0034] Perform 3 rounds of PCR amplification:

[0035] 1. Site-stepping PCR. The PCR system is as follows: a 20 μL reaction system contains 0.5 U of LA Taq enzyme and its buffer, 10 pmol of a syn...

Embodiment 3

[0043] Embodiment 3: Construction of recombinant expression plasmid and recombinant strain of β-agarase gene aga41

[0044] The β-agarase gene aga41 obtained in the present invention is cloned into an expression vector to construct a recombinant expression strain. First, based on the full-length sequence, design the upstream primer aga41F (5'-CACTAC CATATG TACTGTTCGTTTTA-3', NdeI) and downstream primer aga41R (5'-ATTCTCGAGTCATTTGTTAATAGATC-3', XhoI), PCR amplification confirmed the full-length sequence of the gene. The expression plasmid was constructed by enzyme digestion cloning, that is, the PCR product was double-digested with NdeI and XhoI, the digested fragment was recovered by tapping the rubber, and it was ligated with the plasmid pET28b that had also been double-digested with NdeI and XhoI. 2 The transformation method was transformed into Escherichia coli DH5α, and positive clones were screened for kanamycin resistance. A plasmid extraction kit (Axygen) was used to...

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Abstract

The invention discloses an agarase and an acquisition method of an encoding gene thereof. The acquisition method utilizes various PCR methods to acquire the agarase gene aga41 from marine bacteria vibrio natriegens (the preservation number is CGMCC No.2428) for the first time and breaks through the conventional method which screens the agarase gene by constructing a library. The agarase gene aga41 belongs to a GH50 family in a beta-agarase. The whole length of the agarase gene aga41 is 2,973bp. 990 amino acids are encoded on the agarase gene aga41. The highest sequence similarity of the agarase gene aga41 with the existing agarase is 49 percent. The agarase gene aga41 is a novel beta-agarase. An activated recombinant agarase which is obtained by cloning the agarase gene of the invention on an expression vector pET28b, constructing a recombinant escherichia coli Rosetta bacterial strain and carrying out heterologous expression can be applied to the industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a β-agarase coding gene and a method for obtaining the gene. Background technique [0002] Agarase (agarase, EC 3.2.1.81) is a glycoside hydrolase that can degrade agar. It mainly exists in microorganisms, especially marine bacteria. A strain of agarase degrading bacteria was first isolated from seawater by Gran in 1902, and an agarase gene was cloned for the first time by Buttner MJ et al. more than 80 years later, which opened up human's understanding of agarase. The agarases discovered so far form a large family, which can be divided into α- and β-agarases according to their mode of action. α-agarase degrades the α-1,3 glucosidic bonds of agar to generate agar oligosaccharide products with different degrees of polymerization; β-agarase degrades the β-1,4 glucosidic bonds of agar to generate new Agar oligosaccharide products. According to the amino acid sequence similarity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N15/70C12N1/21C12N1/19C12N5/10C12N15/10
Inventor 许学伟王春生吴敏廖丽
Owner SECOND INST OF OCEANOGRAPHY MNR
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