CN107828750B - Kelp GDP fucose synthetase, and coding gene and application thereof - Google Patents

Abstract

The invention relates to a kelp GDP fucose synthetase, a coding gene and application thereof; belongs to the field of biology. The amino acid sequence of the kelp GDP fucose synthetase is shown as SEQ ID NO: 1 is shown. The coding gene is shown as SEQ ID NO: 2, respectively. The invention further provides the application of the gene and the encoded protein thereof in the synthesis of fucoidin.

Description

Kelp GDP fucose synthetase, and coding gene and application thereof

Technical Field

The invention relates to a kelp GDP fucose synthetase, a coding gene and application thereof; belongs to the field of biology.

Background

Kelp is a major cultivated marine plant (algae) species throughout the world. As a marine vegetable, kelp has high nutritive value, and meanwhile, the main economic components such as algin, fucoidin (fucoidan) and the like can be widely applied to various industries as raw materials. The algin has wide application, and is mainly applied to the aspects of food industry, medicine and health, textile industry, scientific research and the like. Fucoidin can be used as a binding agent and a blocking agent of metal ions, has physiological functions and efficacies such as HIV virus resistance, anticoagulation, antithrombotic resistance, tumor resistance and the like, and is an important medicinal substance.

Cloning products to synthesize related genes and verifying the functions of the genes, revealing the relationship between the genes and the products, and assisting in developing variety improvement become one of effective ways for improving the content of economic components in the field of international agricultural breeding; meanwhile, the production of active substances and economic products by using genetic engineering techniques has become the core content of the development of the modern biotechnology industry.

Fucoidan (FS) is a polysaccharide mainly present in the cell walls of brown algae and some marine invertebrates, and consists of L-fucose and sulfate groups, in addition to D-xylose, D-galactose or uronic acid.

The biological sources of fucoidan are only algae of Phaeophyceae, marine invertebrates and microorganisms, such as Laminaria digitata (Saccharina japonica), Sargassum (Sargassum), Azotobacter vinelandii (Azotobacter vinelandii), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Halomonas marina. Brown algae such as kelp are natural sources of algin and fucoidin, and the characteristics of large biomass and high content of the brown algae make the brown algae always be the main raw material for extracting the fucoidin.

Disclosure of Invention

The invention aims to provide a kelp GDP fucose synthetase which is named SjaGFS; the amino acid sequence is shown as SEQ ID NO: 1 is shown.

In the present invention, the kelp GDP fucose synthetase catalyzes GDP-4-keto-6-deoxymannose into GDP-4-keto-6-deoxygalactose, and further catalyzes GDP-4-keto-6-deoxygalactose into GDP-fucose. It is a key step in the conversion of GDP-mannose to GDP-fucose in the kelp.

The second purpose of the invention is to provide a gene for encoding kelp GDP fucose synthetase, wherein the gene is separated from kelp (Saccharina japonica) and has a sequence shown in SEQ ID NO: 2, respectively.

The invention also aims to provide the application of the gene and the protein coded by the gene in the synthesis of fucoidin.

The fourth purpose of the invention is the application of the gene and the protein coded by the gene in improving the characters of economic components.

The invention clones a coding gene of kelp GDP fucose synthetase from kelp by a gene cloning method, and experiments prove that the coded protein has remarkable activity of catalyzing GDP-4-ketone-6-deoxymannose into GDP-4-ketone-6-deoxygalactose and is a key gene of fucoidin. Cloning and analyzing the encoding gene of the kelp GDP fucose synthetase provides gene resources for fucoidin gene engineering and molecular breeding. The invention separates the encoding gene of the kelp GDP fucose synthetase from kelp (Saccharomyces japonica) for the first time, and performs enzyme activity detection on recombinant protein through a eukaryotic expression vector, thereby proving the activity function of the kelp GDP fucose synthetase, and having the application values of fucoidin gene engineering and molecular breeding.

Drawings

FIG. 1 is a schematic diagram of the fucoidan synthesis pathway using GDP-mannose as a precursor.

FIG. 2 is a Western-Blot assay of the expression product of Escherichia coli transformed with the gene encoding kelp GDP fucose synthase.

FIG. 3 is a mass spectrum diagram of the enzyme activity detection of the encoded kelp GDP fucose synthetase SjaGFS prepared by the invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the specific experimental conditions not specified in the examples below are generally according to conventional conditions, as described in the molecular Cloning guidelines (Sambrook J, et al.2008.molecular Cloning: A Laboratory Manual,3rd Ed.), or according to the manufacturer's recommendations.

Example 1: cloning and analysis of full-Length coding region of Gene

The kelp is collected from Shandong province, Rongcheng City, and the collection time is 7 months in 2011. The total RNA of the kelp female gametophyte is extracted by adopting a Trizol method, the CDS sequence of the kelp GFS gene is amplified by adopting a Touchd own PCR technology and a common PCR technology by taking a first strand cDNA reverse transcription of the total RNA of the kelp gametophyte as a template, and amplification primers comprise 2 groups (5'-ATTAGAATTCATGGCGGAGACCAGCGGAACCG-3' and 5'-AATTGCGG CCGCCTACTTGCGGGCGGTC-3'; 5'-TTAATGAATTCATGGCGGAGACCAGCG GAACCG-3' and 5'-TTAATGCGGCCGCCTACTTGCGGGCGGTC-3') to obtain a full-length sequence. After the PCR product is detected by 1% agarose gel electrophoresis, a gel block containing a target band is cut under an ultraviolet lamp, a target fragment is recovered by using an agarose gel recovery kit, and the target fragment is stored at the temperature of minus 20 ℃. The recovered target fragment was connected to the cloning vector pGEx-6p-1 overnight in a metal bath at 16 ℃ and transformed into E.coli competent cells E.coli BL21(DE3), spread on LB solid medium containing 100mg/mL Amp, cultured overnight at 37 ℃, and after screening with blue-white spots, 4-10 positive clones were picked for sequencing. And (3) separating a gene coding the kelp GDP fucose synthetase by sequence comparison of sequencing results, and naming the gene as SjaGFS. The full length of the CD S sequence is 984bp, and the nucleotide sequence is shown as SEQ ID NO: 2, the coding sequence codes 327 amino acids, and ATG is used as an initiation codon and TAG is used as a termination codon.

Example 2: preparation and analysis of SjaGFS encoded protein

Detecting the PCR product of the SjaGFS gene of the kelp by 1% agarose gel electrophoresis, cutting a target strip under an ultraviolet lamp, recovering the agarose gel, carrying out EcoRI and NotI double enzyme digestion on the recovered product SjaGFS and pGEx-6p-1 plasmid, carrying out electrophoresis detection by 1% agarose gel after carrying out metal bath at 37 ℃ for 3-4h, and recovering the product by using an agarose gel recovery kit. The target fragment SjaGFS is connected with the plasmid pGEx-6p-1 at 16 ℃ overnight, and the constructed recombinant plasmid is named as pGEx-6 p-1-SjaGFS.

Transforming the recombinant plasmid into an escherichia coli expression strain BL21, selecting BL21 positive clone, shaking and storing the strain. Recombinants were detected by PCR. The PCR product was detected by electrophoresis on a 1% agarose gel and imaged by an automated gel image analyzer. And (3) selecting electrophoresis to detect the correct clone sequencing of the inserted band, and detecting whether mutation exists or not and whether the frame shift is changed or not.

The BL21 strain to which the target fragment was successfully ligated was cultured with shaking at a ratio of 1:1000 (10ml LB liquid medium +10ul AP +10ul bacterial suspension) and the OD was measured every hour until the OD600nm reached 0.6(3-4 h). The bacterial solution was induced with 0.05mM IPTG at 20 ℃ and 160rpm for 6-8 h. After induction is finished, 2ml of bacterial liquid is taken for centrifugation at 4 ℃ and 12000rpm for 5min, supernatant is discarded, and the tube is placed on absorbent paper in an inverted mode; the pellet was added to 1/2 volumes of 1 XPBS (pH 7.5) pre-cooled (i.e., 1ml of 1 XPBS was added to 2ml of the centrifuged pellet) and mixed well. And (2) crushing the bacterial liquid by an ultrasonic method, collecting the crushed supernatant, filtering the supernatant by a filter membrane of 0.45 mu m, purifying a GST column, putting the collected protein sample into a dialysis bag for dialysis to remove unnecessary ions, and obtaining the recombinant SjaGFS protein, wherein the amino acid sequence of the recombinant SjaGFS protein is shown as SEQ ID NO: 1, expression of the recombinant protein was detected by SDS-PAGE and Western-Blot (FIG. 2).

Example 3: functional verification of SjaGFS encoded protein (kelp GDP fucose synthetase)

GM46D-GFS enzyme activity reaction system: 1mL of 100mM MOPS, pH 7.0; 100mM NaCl; 10mM dithiothreitol; 5mM EDTA; 1mM GDP-mannose (Sigma); 0.4mM NADPH; and 1mM NADP. The reaction was initiated by adding 1.5mg/ml of GM46D2 enzyme, reacted at 37 ℃ for 3 hours, followed by addition of 1.5mg/ml of GFS enzyme (prepared in example 2), adjusting the concentration of NADPH to 1.5mM, reacted at 37 ℃ for 2 hours, and then heated to 100 ℃ for 2 minutes to terminate the reaction.

2 percent of trypsin and 0.5 percent of alkaline protease, the protein in the reaction system is removed by treating at 37 ℃ for 3h, and the salt ions in the reaction system are removed by a dialysis bag with the minimum molecular weight cut-off (Solebao, molecular weight cut-off 500). The dialysate was then injected into the mass spectrometer for detection and the results are shown in figure 3. As can be seen from the mass spectrum, peak 605.38 is the reaction substrate GDP-mannose, and peak 589.45 is the reaction product GDP-fucose.

Example 4 study of enzyme Activity of SjaGFS-encoded protein (kelp GDP fucose synthetase)

The SjaGFS-encoded protein prepared in example 2 was subjected to an enzymatic activity test according to the method disclosed in non-patent document Ren Y, Perepolov A V, Wang H, et al. Biochemical catalysis of GDP-L-enzyme de novo synthesis pathway in funnus Mortierella alpina [ J ]. Biochemical & Biophysical Research Communications,2010,391(4):1663-9. the following results were obtained:

	substrate	Vmax(mM/min)	Kcat/min
				Example 2	GDP-4-keto-6-deoxymannose	0.058±0.003	2925.87±121.46

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

Sequence listing

<110> China oceanic university

<120> kelp GDP fucose synthetase, and coding gene and application thereof

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gacttcaagt tcacccccat gaaggaaggc ttgaagcagg catgcgaatg gttttgcgag 960

aactacgaga ccgcccgcaa gtag 984

Claims (3)

1. A kelp GDP fucose synthetase, wherein the gene of the enzyme is separated from kelp; the amino acid sequence of the enzyme is shown as SEQ ID NO: 1 is shown.

2. A gene encoding the GDP fucose synthetase of kelp as claimed in claim 1, the gene sequence being as shown in SEQ ID NO: 2, respectively.

3. Use of the kelp GDP fucose synthase according to claim 1 or the gene according to claim 2 for the synthesis of fucoidan.

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