CN106148364A - Thallus Laminariae (Thallus Eckloniae) vanadium ion dependent form haloperoxidase gene and encoding proteins thereof - Google Patents

Abstract

The present invention relates to gene engineering technology field, specifically one main laminaria vanadium ion dependent form haloperoxidase gene, the aminoacid sequence of this gene nucleotide series and coded protein is respectively SEQ ID NO:1 19 and SEQ ID NO:20 38.The present invention obtains gene order by transcript profile sequencing technologies binding sequence analytical technology, build prokaryotic expression carrier, by recombiant protein is carried out Enzyme assay it was confirmed it has the highest bromoperoxidase and iodo peroxidase activity, without chloro peroxidase activity.This gene has important using value for improving the content of iodine in the algae such as Thallus Laminariae (Thallus Eckloniae).

Description

Vanadium-dependent haloperoxidase gene and its encoded protein in Laminaria

technical field

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

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 extremely important large-scale economic seaweed. It has become a widely planted industrial raw material because it can produce chemical products such as iodine and alginate. At the same time, kelp contains a high amount of iodine, which is the best edible vegetable for supplementing iodine, and its nutritional value is very rich. With the development of the medicinal effects of polysaccharides in kelp in recent years, more and more people have paid attention to its medicinal value, which has promoted the further development of kelp aquaculture. Due to the extremely high iodine content in kelp, a large number of researchers have paid attention to it. Some scientists speculate that the reason why kelp is rich in iodine may be due to the particularity of its iodine oxidase, so its halogenation mechanism and halogen oxidase research has become a hot spot.

The halogenation process of organisms mainly depends on the action of halogenases. Halogenases mainly include haloperoxidase and flavin-dependent halogenase, among which vanadium-dependent haloperoxidase (vHPO) is the most studied. According to its ability to oxidize halogen ions, it can be divided into chloroperoxidase, bromoperoxidase and iodoperoxidase. These three enzymes all have the same conserved metal ion binding site in the gene structure, that is, the imidazole ring of a histidine residue, which is used to bind vanadium ions, and its amino acid sequence is similar to that of acid phosphatases. high degree of homology. Currently, vanadium-dependent haloxidases are found in red algae, green algae and brown algae; however, vanadium-dependent iodoperoxidases are only found in brown algae, especially some higher groups such as Laminaria.

Cloning products to synthesize related genes and verify their functions, revealing the relationship between genes and products, and assisting in the improvement of varieties have become one of the effective ways to increase the content of economic components in the field of international agricultural breeding; at the same time, the use of genetic engineering technology to produce active substances and economic Products have become the core content of the development of modern biotechnology industry. Currently, the cloning of vanadium-dependent haloperoxidase (vHPO) genes in brown algae is scarce, and its function has not yet been confirmed.

Contents of the invention

Aiming at the technical problems existing in the current prior art, the present invention provides a class of haloperoxidase gene, which can encode a haloperoxidase protein, and the protein has very high bromoperoxidase (bromoperoxidase ) and iodoperoxidase (iodoperoxidase) activity, no chloroperoxidase (chloroperoxidase) activity, the gene and its encoded protein can be used to increase the iodine content in algae.

One of the objects of the present invention is to provide a class of haloperoxidase genes, said genes are all haloperoxidase genes isolated from seaweed (Saccharina japonica), the nucleotide sequence of said gene is as SEQID NO : 1-19.

The second object of the present invention is to provide a kind of protein encoded by said gene, said protein is encoded by the nucleotide sequence shown in SEQ ID NO: 1-19, and its amino acid sequence is shown in SEQ ID NO: 20-38 ; It has very high bromoperoxidase and iodoperoxidase activity, no chloroperoxidase activity.

In the present invention, the haloperoxidase gene and its corresponding encoded amino acid sequence are listed as follows:

The third object of the present invention is the application of the gene and its encoded protein in increasing the iodine content in the algae.

The fourth object of the present invention is the application of the gene and its encoded protein in improving the traits of economic components.

The present invention obtains a series of haloperoxidase genes from the kelp transcriptome by the method of transcriptome sequencing, and obtains the full-length sequence through gene artificial synthesis, and proves that the encoded protein has very high bromide Peroxidase and iodoperoxidase activity, no chloroperoxidase activity. The study of kelp haloperoxidase gene will help to understand the mechanism of bromine, iodine and other halogen accumulation in kelp and other brown algae, and also provide genetic resources for genetic engineering and molecular breeding. The present invention obtains the haloperoxidase gene from kelp (Saccharina japonica) for the first time, and expresses the recombinant protein of the gene through the prokaryotic expression vector and detects the enzyme activity, confirming that it has very high bromoperoxidase and iodine Substitute peroxidase activity, no chloroperoxidase activity.

Description of drawings

Fig. 1 is the PCR amplification diagram of the positive clones detected by PCR after the SjvBPO1 gene of the present invention is transformed into the pET32a vector (M is the DNA standard molecular weight).

Fig. 2 is an SDS-PAGE detection chart of the purified expression product of SjvBPO1 gene transformed Escherichia coli BL21 of the present invention.

Fig. 3 is a Western-Blot detection diagram of the expression product of Escherichia coli BL21 transformed with the SjvBPO1 gene of the present invention.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific experimental conditions in the following examples, usually according to conventional conditions, the conditions described in the Molecular Cloning Experiment Guide (Sambrook J, et al.2008. Molecular Cloning: A Laboratory Manual, 3rd Ed.), or according to Conditions recommended by the manufacturer.

Example 1: Preparation of protein encoded by SjvBPO1 sequence

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. The sequencing results were compared to isolate the Laminaria vBPO gene, which was named SjvBPO1. The full length of the SjvBPO1 CDS sequence is 2037bp, and its nucleotide sequence is shown in SEQ ID NO: 1, encoding 678 amino acids, with ATG as the start codon and TAA as the stop codon.

After the kelp SjvBPO1 PCR product was detected by 1% agarose gel electrophoresis, the target band was excised under ultraviolet light, recovered from the agarose gel, and the recovered product SjvBPO1 and pET32a plasmids were digested with EcoRV and NotI, and placed in a metal bath at 37°C After 3-4 hours, it was detected by 1% agarose gel electrophoresis and recovered using an agarose gel recovery kit. The target fragment SjvBPO1 was ligated with the plasmid pET32a, overnight at 16°C, and the constructed recombinant plasmid was named pET32a-SjvBPO1.

The pET32a-SjvBPO1 recombinant plasmid was transformed into Escherichia coli expression strain BL21, and BL21 positive clones were picked, shaken and preserved. PCR detection of recombinants. PCR products were detected by electrophoresis on 1% agarose gel and imaged by automatic gel image analyzer. Pick and sequence the correct clones for electrophoresis detection of the inserted band to detect whether there are mutations and whether the frameshift frame has changed.

The BL21 strain successfully connected with the target fragment was shaken and cultured according to the ratio of 1:1000 (10mL LB liquid medium + 10uL AP + 10uL bacterial liquid), and the OD value was measured every hour until the OD600nm value reached 0.6 (3- 4h). The bacterial solution was induced with a concentration of 0.1 mM IPTG, and the induction condition was 25° C., 160 rpm, for 6-8 hours. After induction, take 2 mL of the bacterial solution and centrifuge at 12,000 rpm at 4°C for 5 min, discard the supernatant, and place the tube upside down on absorbent paper; add 1/2 volume of the precipitate to 1×PBS (PH=7.4) that has been pre-cooled in advance (that is, 2 mL of centrifuged Add 1 mL of 1×PBS to the precipitate, and mix well. Ultrasonic method disrupts the bacterial liquid, collects the crushed supernatant, filters it with a 0.45 μm filter membrane, and purifies it with a Ni column, puts the collected protein sample into a dialysis bag for dialysis to remove unnecessary ions, and obtains the recombinant SjvBPO1 protein, Its amino acid sequence is shown in SEQ ID NO: 20, and the expression of the recombinant protein is detected by SDS-PAGE and Western-Blot.

Example 2

According to the method of Example 1, the protein encoded by SjvBPO2-SjvBPO19 (nucleotide sequence shown in SEQ ID NO: 2-19) was prepared, and the sequence of the prepared protein was shown in SEQ ID NO: 21-38 after sequencing, details as follows:

Example 3: Functional verification of protein encoded by SjvBPO1-19

The detection of SjvBPO1 protein activity adopts the standard method of monochlorodimedone (MCD). The enzyme activity detection reaction was as follows: SjvBPO1 protein was diluted to 0.1 mM with PBS (diluted from 100 microliters to 500 microliters), added 56 microliters of 10 mM Na ₃ VO ₄ , and 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 SjvBPO1 protein, adding 1mM H ₂ O ₂ to start the reaction. Mix the above H ₂ O ₂ removal system and incubate at the corresponding temperature for 2 minutes to start the reaction. Use the corresponding buffer solution as the blank control, and measure the content of MCD in the liquid phase at 278 nm by high performance liquid chromatography. Each reaction was set up with 4 parallel samples. After testing, the enzyme activity using Br- is ^5861.1U /mg, the optimum reaction temperature is 20°C, and the optimum pH is 6.5. Utilizing I ^- enzyme activity is 2342.9U/mg, optimum reaction temperature is 25°C, and optimum pH is 6.5. No enzymatic activity on Cl ^- . The enzyme is a high temperature enzyme, basic protein.

In addition, proteins with similar sequences to SjvBPO1 were detected, but their enzymatic activities were far less than those of the SjvBPO recombinant protein prepared by the present invention, and the enzymatic activity values were compared as follows:

Comparative example 1 is the protein sequence disclosed by GenBank accession number CAD37192.1

Comparative example 2 is the protein sequence disclosed by GenBank accession number CAQ51441.1

Although specific examples have been described herein, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention.

Claims (4)

1. A gene encoding vanadium ion-dependent haloperoxidase in kelp, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1-19. 2. A protein encoded by the gene of claim 1, characterized in that the amino acid sequence of the protein is as shown in SEQID NO: 20-38, which has very high levels of bromoperoxidase and iodoperoxidase Oxidase activity, no chloroperoxidase activity. 3. the application of the enzyme gene described in claim 1 in improving the iodine content in algal body. 4. The application of the protein according to claim 2 in improving the economic composition traits.