Vanadium-dependent haloperoxidase gene and its encoded protein in Laminaria

A technology of peroxidase and vanadium ions, which is applied in the fields of oxidoreductase, plant genetic improvement, genetic engineering, etc. It can solve the problems of lack of vanadium-dependent haloperoxidase clones and unconfirmed functions, and achieve high iodine The effect of substituting peroxidase activity

Active Publication Date: 2019-08-02
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, cloning of vanadium-dependent haloperoxidase (vHPO) genes in brown algae is scarce, and its function remains unidentified

Method used

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  • Vanadium-dependent haloperoxidase gene and its encoded protein in Laminaria
  • Vanadium-dependent haloperoxidase gene and its encoded protein in Laminaria
  • Vanadium-dependent haloperoxidase gene and its encoded protein in Laminaria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of protein encoded by SjvIPO1 sequence

[0016] The full-length sequence of SEQ ID NO:1 was synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd., and the recovered target fragment was connected to the cloning vector pMD19-T in a metal bath at 16°C overnight, and transformed into E. coli competent cells E.coli Top10 , spread on LB solid medium containing 100mg / mL Amp, culture overnight at 37°C, and select 4-10 positive clones for sequencing after IPTG / X-gal blue-white screening. Sequencing results were compared to isolate the HPO gene of Laminaria, named SjvIPO1. The full length of the SjvIPO1 CDS sequence is 1905bp, and its nucleotide sequence is shown in SEQ ID NO: 1, encoding 634 amino acids, with ATG as the start codon and TAA as the stop codon.

[0017] After the kelp SjvIPO1 PCR product was detected by 1% agarose gel electrophoresis, the target band was excised under ultraviolet light, recovered from the agarose gel, and the re...

Embodiment 2

[0021] According to the method of Example 1, the protein encoded by SjvIPO2-SjvIPO37 (nucleotide sequence shown in SEQ ID NO: 2-37) was prepared, and the sequence of the prepared protein was shown in SEQ ID NO: 39-74 after sequencing, details as follows:

[0022]

[0023]

Embodiment 3

[0024] Example 3: Functional verification of SjvIPO1-37 encoded protein

[0025] The detection of SjvIPO1 protein activity adopts the standard method of monochlorodimedone (MCD). The enzyme activity detection reaction is as follows: Dilute the SjvIPO1 protein to 0.1 mM with PBS (dilute 100 microliters to 500 microliters), add 56 microliters of 10 mM Na 3 VO 4 , mixed at low speed overnight. 50mM MES (pH 6.50), 200mM KBr (or KCl, KI), 0.1mM MCD (monochlorodimedone) and appropriate amount of Na 3 VO 4 Treated recombinant SjvIPO1 protein, add 1mM H 2 o 2 initial reaction. Remove the above H 2 o 2 The system was mixed and incubated at the corresponding temperature for 2 minutes before starting the reaction. Using the corresponding buffer as a blank control, the content of MCD in the liquid phase was measured at 278 nm by high performance liquid chromatography, and 4 parallel samples were set for each reaction. . After testing, using I - The enzyme activity is 5895U / mg, ...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to kelp vanadium ion dependent halogen peroxidase gene, and nucleotide sequences and encoding protein amino acid sequences of the gene are respectively SEQ ID NO: 1-37 and SEQ ID NO: 38-74. Gene sequences are obtained by the combination of transcriptome sequencing technology and sequence analysis technology to construct prokaryotic expression vectors, and by subjecting recombinant proteins to enzymatic activity detection, it is proved that the proteins have very high iodo-peroxidase activity and have no bromo-peroxidase and chloro-peroxidase activity. The gene has important application value for increasing iodine content in algae such as kelp.

Description

technical field [0001] The invention relates to a class of coding enzyme gene of kelp haloperoxidase. In particular, it relates to the nucleotide sequence of kelp (Saccharinajaponica) haloperoxidase gene, its coded protein and its application in the halogenation process of kelp and in improving the traits of important economic components. Background technique [0002] A variety of halides have been found in the ocean, most of which have high medicinal and application value. In the ocean, the range of producers that can produce halides is extremely wide, with a wide variety and forms. Among them, marine algae have a strong ability to absorb halogens, and the halogens in their bodies are more abundant. Kelp is the main species of marine plants (algae) cultivated in the world, and its iodine content is extremely high. 10 mg of iodine can be isolated from 1 g of dry weight of kelp, accounting for 1% of its dry weight. Kelp is mainly cultivated in my country. It is an extremel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04
CPCC12N9/0065C12Y111/01806
Inventor 刘涛
Owner OCEAN UNIV OF CHINA
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