Cloning and application of mannuronic acid C5-isomerase gene related to kelp high-temperature response

Abstract

The invention discloses cloning and application of a mannuronic acid C5-isomerase gene related to high-temperature response of kelp, and belongs to the technical field of biology. The mannuronic acid C5-isomerase (MC5E) gene related to high-temperature stress response of the kelp is screened out; on the basis, designing a specific primer, and cloning from kelp to obtain a cDNA (complementary deoxyribonucleic acid) sequence of the MC5E gene; a prokaryotic expression system is utilized, soluble recombinant protein expression is successfully obtained, and high-purity MC5E protein is obtained through purification; through nuclear magnetic resonance hydrogen spectrum analysis and judgment, algin mannuronic acid (M) can be converted into guluronic acid (G) by the recombinant protein, and the content of G in algin is increased; fluorescent quantitative PCR detection shows that under high temperature stress, the transcriptional level of the kelp juvenile sporophyte MC5E gene is remarkably increased, which indicates that the kelp juvenile sporophyte MC5E gene participates in response of kelp to high temperature stress.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and biotechnology, and relates to the cloning of a mannuronate C5-isomerase gene related to high temperature response of kelp and application thereof. Background Art

[0002] Kelp is one of the main cultivated brown algae in my country and has important economic, social and ecological values. Kelp originated from the coast of Hokkaido, Japan, and is a cold-water subtidal brown algae. Compared with its place of origin, the water temperature in my country's kelp cultivation areas, especially in the southern cultivation areas, is higher, and kelp often suffers from high temperature stress during seedling and cultivation. Rising water temperature will make it difficult to grow vegetables in summer, shorten the growth temperature period, and significantly increase the risk of diseases during the breeding and cultivation of kelp seedlings, limiting the further improvement of kelp production and quality. Therefore, cultivating new high-temperature resistant varieties is of great significance to the healthy and sustainable development of the kelp cultivation industry. Mining genes related to high temperature stress response at the genome and transcriptome levels, functionally analyzing and utilizing key genes can not only promote the analysis of kelp functional genes, but also lay a good theoretical foundation for molecular design breeding.

[0003] Alginic acid is a structural component of the cell wall of brown algae. It exists in the intercellular mucus and cell wall in the form of soluble calcium, magnesium, potassium, and sodium salts, collectively known as alginate. The presence of alginate gives brown algae mechanical rigidity and elasticity, and plays a protective role when brown algae are subjected to environmental stress. Alginic acid (C6H8O6) n It is a polysaccharide formed by linear polymerization of β-D-mannuronic acid (M) and α-L-guluronic acid (G) through β-1,4 glycosidic bonds. In the body, alginate is first synthesized in the form of polymannuronic acid, and then mannuronic acid C5-isomerase (MC5E) can specifically catalyze the conversion of M residues to G residues at the polymer level. Current studies have shown that there are multiple MC5E encoding genes in brown algae, but the functions and regulatory mechanisms of each MC5E need to be further analyzed. Summary of the invention

[0004] The present invention aims to provide a cloning and application of a mannuronate C5-isomerase gene related to high temperature response of Laminaria japonica.

[0005] The present invention screened out a mannuronate C5-isomerase (MC5E) gene related to high temperature stress response; on this basis, specific primers were designed to clone the gene from Laminaria japonica. MC5EThe cDNA sequence of the gene was obtained; the soluble recombinant protein expression was successfully obtained by using the prokaryotic expression system, and the high-purity MC5E protein was purified; the nuclear magnetic resonance hydrogen spectrum analysis showed that the recombinant protein could convert alginate mannuronic acid (M) into guluronic acid (G), thereby increasing the G content in alginate; the fluorescence quantitative PCR test showed that under high temperature stress, the MC5E The transcription level of the gene increased significantly, indicating that it is involved in the response of kelp to high temperature stress.

[0006] The technical solution of the present invention is as follows: The present invention provides a mannuronate C5-isomerase gene, the nucleotide sequence of the gene is shown as SEQ ID NO.1 in the sequence table.

[0007] The protein encoded by the laminaria mannuronate C5-isomerase gene has the amino acid sequence in the sequence table SED ID NO.2.

[0008] The invention provides a method for cloning a Laminaria japonica mannuronate C5-isomerase (MC5E) gene.

[0009] The invention provides a recombinant expression plasmid containing a Laminaria japonica mannuronate C5-isomerase (MC5E) gene.

[0010] The invention provides a recombinant genetic engineering strain containing a Laminaria japonica mannuronate C5-isomerase (MC5E) gene.

[0011] The nucleotide and amino acid sequences of the Laminaria japonica mannuronate C5-isomerase of the present invention can also be obtained by artificial synthesis based on the predicted sequences.

[0012] The present invention also provides the application of the protein encoded by the Laminaria japonica mannuronic acid C5-isomerase gene, which can be specifically used for catalyzing the conversion of mannuronic acid to guluronic acid.

[0013] The enzyme preparation prepared by using the encoded protein can increase the content of guluronic acid (G) in alginate and reduce the M / G ratio of alginate.

[0014] Furthermore, the present invention also provides the application of the mannuronate C5-isomerase gene in kelp breeding, and further provides the application of the mannuronate C5-isomerase gene in cultivating high temperature resistant kelp varieties.

[0015] Beneficial effects of the present invention: The present invention screened out a mannuronate C5-isomerase (MC5E) gene related to high temperature stress response; on this basis, specific primers were designed to clone the gene from Laminaria japonica. MC5EThe cDNA sequence of the gene was obtained; the soluble recombinant protein expression was successfully obtained by using the prokaryotic expression system, and the high-purity MC5E protein was purified; the nuclear magnetic resonance hydrogen spectrum analysis showed that the recombinant protein could convert alginate mannuronic acid (M) into guluronic acid (G), thereby increasing the G content in alginate; the fluorescence quantitative PCR test showed that under high temperature stress, the MC5E The transcription level of the gene increased significantly, indicating that it is involved in the response of Laminaria japonica to high temperature stress. The functional analysis and utilization of the Laminaria japonica mannuronate C5-isomerase gene of the present invention can not only promote the analysis of Laminaria japonica functional genes, but also lay a good theoretical foundation for the in vitro modification of alginate and the molecular design breeding of Laminaria japonica. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The cloning result of the gene provided in the embodiment of the present invention. MC5E a, electrophoresis of gene amplification; b, electrophoresis of bacterial liquid detection.

[0017] Figure 2 This is the result of multiple sequence alignment between the protein sequence provided in the embodiment of the present invention and homologous genes of other species.

[0018] Figure 3 This is the WB test result of the purified mannuronate C5-isomerase protein provided in the embodiment of the present invention.

[0019] Figure 4 The effect of mannuronic acid C5-isomerase on alginic acid provided by the embodiment of the present invention 1 H-NMR spectrum.

[0020] Figure 5 The embodiment of the present invention provides MC5E Gene expression patterns in Laminaria japonica under high temperature stress conditions. DETAILED DESCRIPTION

[0021] The features and advantages of the present invention can be further understood by the following detailed description. The examples provided are only illustrative of the methods of the present invention and are not intended to limit the remainder of the present invention in any way. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] The present invention is further described in the following examples, but the present invention is not limited thereto. Example 1

[0023] The kelp of the present invention MC5E The gene cloning method mainly includes the following steps: 1. RNA extraction and cDNA synthesis from Laminaria japonica; 2. MC5E Gene PCR amplification and product sequencing; 3. MC5E protein structure prediction and analysis.

[0024] The specific operations are as follows: 1. Extraction of RNA from Laminaria japonica: Grind 40 mg of Laminaria japonica female gametophyte sample in a mortar pre-cooled with liquid nitrogen, and extract total RNA from Laminaria japonica female gametophyte according to the steps of the Plant RNA Kit. Evo M-MLV PluscDNA Synthesis Kit synthesizes first-strand cDNA for gene amplification.

[0025] 2. MC5E Gene PCR amplification and product sequencing: Designed using NCBI online MC5E Gene-specific primers MC5E-F (5'-CTCACTCTCCGCACCACAAT-3') and MC5E-R (5'-GCACATCGTGTCCAGACCAA-3'). The amplification system was: 1 μl cDNA, 2 μl MC5E-F, 2 μl MC5E-R, 10 μl Phanta Max Master Mix (2×), 5 μl ddH2O. The reaction procedure was: 95°C pre-denaturation for 3 min; then 35 cycles: 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 2 min; and finally 72°C extension for 5 min. After DNA electrophoresis detection, 0.5ul of the PCR product was directly aspirated and connected to the pBlunt-Zero cloning vector, transformed into Fast-F1 competent cells, and amplified and detected using universal M13 primers. The positive bacterial solution was taken for sequencing verification (see Figure 1 ).

[0026] 3. MC5E protein structure prediction analysis: prediction using ORF finder tool MC5E The open reading frame of the gene was identified, and the molecular weight (MW) and isoelectric point (pI) of the protein were predicted using the ProtParam online tool. The signal peptide sequence of the protein was predicted using SignalP 5.0, and whether the protein contained a transmembrane structure was predicted using TMHMM Server v. 2.0. Homologous sequences were downloaded from the NCBI website, multiple sequence alignment was performed using ClustalX, and mapping was performed using the ESPript 3.0 tool. The sequencing results showed that the amplified MC5EThe gene sequence is 1843 bp long, of which the CDS sequence is 1494 bp long, encoding 497 aa sequences (sequence see SEDIQ No.1), the molecular weight of the encoded protein is 53.97 kDa, and the theoretical isoelectric point is 4.44. It is predicted that MC5E contains a signal peptide sequence, the cleavage site is between the 21st and 22nd amino acids, and does not contain a transmembrane structure (sequence see SEDIQ No.2). The results of multiple sequence alignment show that the MC5E described in the present invention has a conserved catalytic domain YG(F / I)DPH(D / E) of this family gene (see Figure 2 ).

[0027] SED ID No.1 Laminaria japonica mannuronate C5-isomerase (MC5E) gene base sequence: SED ID No.2 Laminaria japonica mannuronate C5-isomerase (MC5E) gene encoding protein sequence: MSKALGKTALMSMLALKLAAAADTASPNDPIPKPKCDSSQMVSIRYSSASARLYLESGDG STRGGCVTLQQIWENRGGKEPLYAVDPDSGDISDTATGTWLLTEELFVEDGITLQVHGSS AGGDADELRLLSTKDTFINLRAHGGSLDFLSTKVFSWDSSTNGPDEDETDGRSYISAVSE KLDGQTCLGRAKKEMGEARMDIEDSEMGYLGYHDSESYGLTWKVRGFCTDKSNPEVFDDSNVYGNIYDSDIHHMNFGVYTYGHQQGDWRRNKMHDNSGYGFDPHDDSDFLTIHDNEVYNNGYHGIIASKRCNGVSIQGNEVYGGAETSAGIFLHRSSD DAIVKGNYVHDNGDAGLAMLESFNADVSDNTFENNKYGVRFSVGCGDNVFTNNLISGSSKYNTFSYLGSDAPDVVDSGRSQNNIFQDNTIVGGEESIKLKESDGTHFIDNKFEDVTTIRFDDATGIVMSGNTGLDDVELKVANGACFDEASDSDFTPTC Example 2

[0028] This embodiment provides a method for expressing, purifying and determining the enzyme activity of Laminaria japonica MC5E recombinant protein, which mainly includes the following steps: 1. Expression of MC5E recombinant protein; 2. Purification of MC5E recombinant protein; 3. Analysis of MC5E recombinant protease activity.

[0029] The specific operations are as follows: 1. Expression of MC5E recombinant protein: According to the amplified sequence, the coding region was codon optimized and then the gene was synthesized and connected to the pCold Ⅱ expression vector. N I and EcoRThe restriction sites were separated and transformed into TOP10 competent cells. The strains that were positive after sequencing were expanded, plasmids were extracted, transformed into BL21 (DE3) competent cells, and sequenced for verification. Take an appropriate amount of bacterial liquid and culture it overnight at 37°C. The next day, add it to LB medium (Amp + ), when OD600 was about 0.6, 0.1 mM IPTG was added and induced at 15°C and 160 rpm for 24 h.

[0030] 2. Purification of MC5E recombinant protein: The bacterial solution was centrifuged at 8000 rpm and 4°C for 15 min, the bacterial precipitate was collected, resuspended in a buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 4mM CaCl2, 40 mM imidazole), and ultrasonically broken in an ice bath until clear. Centrifuged at 4°C and 13000 rpm for 40 min, the supernatant obtained by centrifugation was filtered with a 0.22 μm filter membrane. The recombinant protein was subjected to affinity chromatography using an AKTA pure system and a His Trip HP prepacked column. The protein was linearly eluted using an elution buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 4mM CaCl2, 500 mM imidazole). The protein obtained by affinity chromatography was subjected to gel filtration chromatography, the buffer composition: 20 mM HEPES (pH 7.4); 500 mM NaCl, 4mM CaCl2. Protein purity was checked by WB (see Figure 3 ).

[0031] 3. Analysis of MC5E recombinant protease activity: Prepare 10 ml of reaction solution according to the following reaction system: 10 mM HEPES, 0.1 M NaCl, 10 μg MC5E, 0.1% alginate. At the same time, prepare a control reaction system without MC5E. Incubate the reaction solution in a 30°C water bath for 30 h. After the reaction, the reaction solution was heated at 95°C for 10 min and centrifuged at 12,000 rpm to remove denatured proteins; 2 volumes of anhydrous ethanol were added to the supernatant for precipitation twice; the precipitate was dissolved in 10 ml ultrapure water, the pH was adjusted to pH 5.6 with HCl, and placed in a 100°C water bath for 1 h; the pH was adjusted to pH 3.8 with HCl, and the sample was placed in a 100°C water bath for 30 min; the pH was adjusted to pH 7-8 with NaOH, and the sample was freeze-dried; the sample was dissolved in 5 ml D2O and freeze-dried again; finally, the sample was dissolved in 700 ul D2O, 20 ul 0.3 M TTHA chelating agent was added, and the spectrum of the sample was detected on a nuclear magnetic resonance hydrogen spectrometer (Bruker Avance III 600 MHz) at a detection temperature of 80°C ( Figure 4 ). The relative frequency of each block is calculated according to the international standard (F2259-10 (2012)). The results are as follows Figure 4 As shown in Table 1: Compared with the control group, the experimental group F MM , F MG , F GM reduce, F GG The M / G ratio decreased from 1.48 before the reaction to 1.01, indicating that MC5E can play the catalytic activity of mannuronate C5-isomerase. MC5E The protein encoded by the gene can be used as an enzyme preparation to increase the content of guluronic acid (G) in alginate.

[0032] Table 1 Example 3

[0033] This embodiment provides a kelp MC5E Analysis of gene expression patterns under high temperature stress mainly includes the following steps: 1. High temperature stress treatment experiment; 2. RNA extraction and cDNA synthesis from Laminaria japonica; 3.qRT-PCR analysis.

[0034] The specific operations are as follows: 1. High temperature stress treatment experiment: Young kelp sporophytes with a length of 20-30 cm were collected from the culture raft in Rongcheng City, Shandong Province. After the kelp samples were rinsed, they were incubated at a temperature of 25°C and a light intensity of 35±5 μmol / m 2 / s and cultured under a photoperiod of 12:12 (L:D). Samples were taken at 0, 1, 3, 6, 12, and 24 h, respectively. All samples were wiped dry and quickly frozen in liquid nitrogen and stored at -80°C.

[0035] 2. Extraction of RNA from Laminaria japonica and synthesis of cDNA: The RNA from Laminaria japonica was extracted by referring to the extraction method described in Example 1 of the present invention. Evo M-MLV Mix Kit with gDNA Clean for qPCR synthesizes first-strand cDNA for qRT-PCR detection.

[0036] 3.qRT-PCR analysis: Actin (F: 5'-GACGGGTAAGGAAGAACGG-3'; R: 5'-GGGACAACCAAAACAAGGGCAGGAT-3') was used as the internal reference gene to design the MC5EGene-specific primers qMC5E-F (5′-CATCGAGGACAGCGAGATGG-3′) and qMC5E-R (5′-ACCCCGAAGTTCATGTGGTG-3′) were used for quantitative experiments.

[0037] 4. Prepare the reaction system according to the instructions of 2×SYBR Green qPCR Mix (With ROX). The reaction procedure is: 94°C pre-denaturation for 3 min; then enter 40 cycles: 94°C denaturation for 10 s, 55°C annealing for 10 s, and 72°C extension for 30 s. Repeat four times for each template setting, using 2 -△△Ct The results show that with the extension of high temperature treatment time, the MC5E The gene transcription level showed an increasing trend. When treated with high temperature for 3 h, 6 h, 12 h and 24 h, the gene transcription level was 1.10, 8.70, 7.58 and 34.02 times that of the control group, respectively. Figure 5 This indicates that the gene is involved in the response of Laminaria japonica to high temperature stress. MC5E The gene can be used in the screening or identification of high temperature resistant germplasm.

[0038] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. A mannuronate C5-isomerase gene, characterized in that: The nucleotide sequence of the gene is shown as SEQ ID NO.1 in the sequence listing.

2. The protein encoded by the Laminaria japonica mannuronate C5-isomerase gene according to claim 1, characterized in that: The encoded protein has the amino acid sequence in the sequence listing SED ID NO.

2.

3. Use of the mannuronate C5-isomerase gene according to claim 1 in kelp breeding.

4. Use of the mannuronate C5-isomerase gene according to claim 1 in cultivating high temperature resistant kelp varieties.

5. Use of the encoded protein according to claim 2 in catalyzing the conversion of mannuronic acid to guluronic acid.

6. Use of the encoded protease preparation according to claim 2 in increasing the content of guluronic acid (G) in alginate and reducing the M / G ratio of alginate.

7. A method for cloning a mannuronate C5-isomerase gene, comprising the following steps:

1. RNA extraction and cDNA synthesis from Laminaria japonica; 2. MC5E Gene PCR amplification and product sequencing; 3. MC5E protein structure prediction and analysis.

8. A mannuronate C5-isomerase gene expression vector, characterized in that: An expression vector containing the mannuronate C5-isomerase gene according to claim 1 as a target gene.

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