Cloning and application of a gene related to high temperature response of mannuronate c5-epimerase in laminaria

Abstract

This invention discloses the cloning and application of a mannuronic acid C5-isomerase gene related to high-temperature response in kelp, belonging to the field of biotechnology. This invention screens out a mannuronic acid C5-isomerase (MC5E) gene related to high-temperature stress response in kelp; based on this, specific primers are designed to clone the gene from kelp. MC5E The cDNA sequence of the gene was obtained; soluble recombinant protein was successfully expressed using a prokaryotic expression system, and high-purity MC5E protein was purified; 1H NMR spectroscopy analysis showed that this recombinant protein can convert mannuronic acid (M) in alginate to guluronic acid (G), increasing the G content in alginate; quantitative real-time PCR detection showed that under high temperature stress, kelp larvae... MC5E The significantly elevated transcription level of the gene indicates its involvement in kelp's response to high-temperature stress.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biotechnology, and relates to the cloning and application of the mannuronic acid C5-isomerase gene related to the high-temperature response of kelp. Background Technology

[0002] Kelp is one of the main cultivated brown algae in my country, possessing significant economic, social, and ecological value. Originating from the coast of Hokkaido, Japan, kelp is a cold-water subtidal brown algae. Compared to its native habitat, the water temperatures in my country's kelp cultivation areas, especially in the south, are higher, frequently subjecting kelp to high-temperature stress during seedling and growth. Increased water temperatures make it difficult for kelp to survive the summer, shorten the optimal growth period, and significantly increase the risk of diseases during seedling propagation and cultivation, limiting further improvements in kelp yield and quality. Therefore, cultivating new heat-resistant varieties is crucial for the healthy and sustainable development of the kelp aquaculture industry. Identifying genes related to high-temperature stress responses at the genomic and transcriptomic levels, and performing functional analysis and utilization of key genes, can not only advance the understanding of kelp functional genes but also lay a solid 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 as 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 they are subjected to environmental stress. Alginic acid (C6H8O6) n Alginic acid is a polysaccharide formed by the linear polymerization of β-D-mannuronic acid (M) and α-L-guluronic acid (G) via β-1,4 glycosidic bonds. In organisms, alginate is initially synthesized as polymannuronic acid, and subsequently, the mannuronic acid C5-isomerase (MC5E) specifically catalyzes the conversion of M residues to G residues at the polymer level. Current research indicates the existence of multiple MC5E-encoding genes in brown algae, but the functions and regulatory mechanisms of each MC5E require further elucidation. Summary of the Invention

[0004] The purpose of this invention is to provide a clone of the mannouronic acid C5-isomerase gene related to the high-temperature response of kelp and its application.

[0005] This invention screened a mannouronic acid C5-isomerase (MC5E) gene associated with high-temperature stress response; based on this, specific primers were designed to clone the gene from kelp. MC5EThe cDNA sequence of the gene was obtained; soluble recombinant protein was successfully expressed using a prokaryotic expression system, and high-purity MC5E protein was purified; 1H NMR spectroscopy analysis showed that this recombinant protein can convert mannuronic acid (M) in alginate to guluronic acid (G), increasing the G content in alginate; quantitative real-time PCR detection showed that under high-temperature stress, this... MC5E The significantly elevated transcription level of the gene indicates its involvement in kelp's response to high-temperature stress.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a mannouronic acid C5-isomerase gene, the nucleotide sequence of which is shown in SEQ ID NO.1 in the sequence listing.

[0008] The protein encoded by the kelp mannouronic acid C5-isomerase gene has the amino acid sequence listed in SED ID NO.2.

[0009] This invention provides a method for cloning the kelp mannouronic acid C5-isomerase (MC5E) gene.

[0010] This invention provides a recombinant expression plasmid containing the kelp mannouronic acid C5-isomerase (MC5E) gene.

[0011] This invention provides a recombinant genetically engineered strain containing the kelp mannouronic acid C5-isomerase (MC5E) gene.

[0012] The nucleotide and amino acid sequences of the kelp mannouronic acid C5-isomerase of the present invention can also be artificially synthesized based on the predicted sequences.

[0013] The present invention also provides the application of the protein encoded by the kelp mannuronic acid C5-isomerase gene, specifically, it can be used to catalyze the conversion of mannuronic acid to guluronic acid.

[0014] Enzyme preparations made using encoded proteins can increase the content of guluronic acid (G) in alginate and reduce the M / G ratio of alginate.

[0015] Furthermore, this invention also provides the application of the described mannulate C5-isomerase gene in kelp breeding. Even further, it provides its application in cultivating heat-resistant kelp varieties.

[0016] The beneficial effects of this invention are:

[0017] This invention screened a mannouronic acid C5-isomerase (MC5E) gene associated with high-temperature stress response; based on this, specific primers were designed to clone the gene from kelp. MC5EThe cDNA sequence of the gene was obtained; soluble recombinant protein was successfully expressed using a prokaryotic expression system, and high-purity MC5E protein was purified; 1H NMR spectroscopy analysis showed that this recombinant protein can convert mannuronic acid (M) in alginate to guluronic acid (G), increasing the G content in alginate; quantitative real-time PCR detection showed that under high-temperature stress, this... MC5E The significantly elevated transcription level of the gene indicates its involvement in kelp's response to high-temperature stress. This invention's functional analysis and utilization of the kelp mannouronic acid C5-isomerase gene not only advances the understanding of kelp functional genes but also lays a solid theoretical foundation for the in vitro modification of alginate and molecular design breeding of kelp. Attached Figure Description

[0018] Figure 1 The cloning results of the gene provided in the embodiments of the present invention. Wherein, a, MC5E a) Gene amplification electrophoresis image; b) Bacterial culture detection electrophoresis image.

[0019] Figure 2 The results of comparing the protein sequence provided in the embodiments of the present invention with the homologous gene multiple sequence of other species.

[0020] Figure 3 The WB detection results of the purified mannulac C5-isomerase protein provided in the embodiments of the present invention.

[0021] Figure 4 The effect of mannuronic acid C5-isomerase on alginate provided in this embodiment of the invention 1 H-NMR spectrum.

[0022] Figure 5 Provided for embodiments of the present invention MC5E Gene expression patterns under high-temperature stress conditions in kelp. Detailed Implementation

[0023] The features and advantages of the present invention will be further understood through the following detailed description. The provided embodiments are merely illustrative of the methods of the present invention and do not limit the remaining content disclosed herein in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0024] The invention will be further illustrated in the following embodiments, but the invention is not limited thereto. Example 1

[0025] The kelp described in this invention MC5E Gene cloning methods mainly include the following steps:

[0026] 1. Kelp RNA extraction and cDNA synthesis;

[0027] 2. MC5E Gene PCR amplification and product sequencing;

[0028] 3. Predictive analysis of MC5E protein structure.

[0029] The specific steps are as follows:

[0030] 1. Extraction of RNA from kelp: 40 mg of female kelp gametophyte sample was ground in a mortar pre-cooled with liquid nitrogen, and total RNA was extracted from the female kelp gametophyte following the steps of the Plant RNA Extraction Kit. Using... Evo M-MLV The PluscDNA Synthesis Kit synthesizes first-strand cDNA for gene amplification.

[0031] 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') were used. The amplification system consisted of 1 μl cDNA, 2 μl MC5E-F, 2 μl MC5E-R, 10 μl Phanta Max Master Mix (2×), and 5 μl ddH2O. The reaction program was as follows: 95°C pre-denaturation for 3 min; followed by 35 cycles: 95°C denaturation for 15 s, 56°C annealing for 15 s, and 72°C extension for 2 min; and finally, 72°C extension for 5 min. After DNA electrophoresis, 0.5 μl of the PCR product was directly ligated into the pBlunt-Zero cloning vector, transformed into Fast-F1 competent cells, and amplified using universal M13 primers. Positive bacterial cultures were then sequenced for verification (see [link to relevant documentation]). Figure 1 ).

[0032] 3. MC5E protein structure prediction analysis: prediction using the ORF finder tool. MC5E The open reading frame of the gene was determined, and the molecular weight (MW) and isoelectric point (pI) of the protein were predicted using the online tool ProtParam. The signal peptide sequence of the protein was predicted using SignalP 5.0, and the presence of transmembrane structures was predicted using TMHMM Server v. 2.0. Homologous sequences were downloaded from the NCBI website, and multiple sequence alignment was performed using ClustalX. Graphing was then performed using ESPript 3.0. Sequencing results showed that the amplified protein obtained according to the present invention... MC5EThe gene sequence is 1843 bp long, of which the CDS sequence is 1494 bp long, encoding 497 amino acid sequences (sequence details in SEDIQ No. 1). The encoded protein has a molecular weight (MW) of 53.97 kDa and a theoretical isoelectric point of 4.44. Prediction indicates that MC5E contains a signal peptide sequence with a cleavage site between amino acids 21 and 22, and does not contain transmembrane structures (sequence details in SEDIQ No. 2). Multiple sequence alignment results show that the MC5E described in this invention contains a conserved catalytic domain YG(F / I)DPH(D / E) of this gene family (see...). Figure 2 ).

[0033] SED ID No. 1 Kelp mannouronic acid C5-isomerase (MC5E) gene base sequence:

[0034]

[0035] The protein sequence encoded by the kelp mannouronic acid C5-isomerase (MC5E) gene, SED ID No. 2:

[0036] MSKALGKTALMSMLALKLAAAADTASPNDPIPKPKCDSSQMVSIRYSSASARLYLESGDG

[0037] STRGGCVTLQQIWENRGGKEPLYAVDPDSGDISDTATGTWLLTEELFVEDGITLQVHGSS

[0038] AGGDADELRLLSTKDTFINLRAHGGSLDFLSTKVFSWDSSTNGPDEDETDGRSYISAVSE

[0039] KLDGQTCLGRAKKEMGEARMDIEDSEMGYLGYHDSESYGLTWKVRGFCTDKSNPEVFDDSNVYGNIYDSDIHHMNFGVYTYGHQQGDWRRNKMHDNSGYGFDPHDDSDFLTIHDNEVYNNGYHGIIASKRCNGVSIQGNEVYGGAETSAGIFLHRSSD DAIVKGNYVHDNGDAGLAMLESFNADVSDNTFENNKYGVRFSVGCGDNVFTNNLISGSSKYNTFSYLGSDAPDVVDSGRSQNNIFQDNTIVGGEESIKLKESDGTHFIDNKFEDVTTIRFDDATGIVMSGNTGLDDVELKVANGACFDEASDSDFTPTC Example 2

[0040] This embodiment provides a method for the expression, purification, and enzyme activity determination of kelp MC5E recombinant protein, mainly including the following steps:

[0041] 1. Expression of recombinant MC5E protein;

[0042] 2. Purification of MC5E recombinant protein;

[0043] 3. MC5E recombinant protease activity analysis.

[0044] The specific steps are as follows:

[0045] 1. MC5E recombinant protein expression: Based on the amplified sequence, the coding region was codon-optimized to synthesize the gene, which was then ligated into the pCold II expression vector. Nde I and EcoR The restriction enzyme sites were identified and the culture was transformed into TOP10 competent cells. Strains that tested positive for sequencing were expanded, plasmids were extracted, transformed into BL21(DE3) competent cells, and sequenced for verification. An appropriate amount of bacterial culture was incubated overnight at 37°C, and the next day, it was added to LB medium (Amp) at a ratio of 1:100. + When the OD600 is about 0.6, 0.1 mM IPTG is added, and the mixture is induced for 24 h at a temperature of 15℃ and a rotation speed of 160 rpm.

[0046] 2. Purification of MC5E recombinant protein: The bacterial culture was centrifuged at 8000 rpm for 15 min at 4°C, and the bacterial pellet was collected and resuspended in buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 4 mM CaCl2, 40 mM imidazole). The pellet was then sonicated until clear under ice bath conditions. The pellet was centrifuged at 13000 rpm for 40 min at 4°C, and the supernatant was filtered through a 0.22 μm filter. Affinity chromatography of the recombinant protein was performed using an AKTA pure system and a His Trip HP pre-packed column. The protein was linearly eluted using elution buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 4 mM CaCl2, 500 mM imidazole). The protein obtained from affinity chromatography was then subjected to gel filtration chromatography with the following buffer composition: 20 mM HEPES (pH 7.4); 500 mM NaCl; 4 mM CaCl2. Protein purity was determined using Western blotting (see...) Figure 3 ).

[0047] 3. MC5E recombinant protease activity analysis: Prepare 10 ml of reaction solution according to the following reaction system: 10 mM HEPES, 0.1 M NaCl, 10 μg MC5E, and 0.1% alginate. A control reaction system without MC5E was also prepared. Incubate the reaction solution in a 30°C water bath for 30 h. After the reaction, the reaction solution was heated at 95℃ for 10 min and centrifuged at 12000 rpm to remove denatured proteins. Two volumes of anhydrous ethanol were added to the supernatant to precipitate twice. The precipitate was dissolved in 10 ml of ultrapure water, and the pH was adjusted to pH 5.6 with HCl and placed in a 100℃ water bath for 1 h. The pH was then adjusted to pH 3.8 with HCl, and the sample was placed in a 100℃ water bath for 30 min. The pH was adjusted to pH 7-8 with NaOH, and the sample was freeze-dried. The sample was then dissolved again in 5 ml of D2O and freeze-dried. Finally, the sample was dissolved in 700 μL of D2O, and 20 μL of 0.3 M TTHA chelating agent was added. The sample spectrum was detected on a 1H NMR spectrometer (Bruker Avance III 600 MHz) at 80℃. Figure 4 The relative frequencies of each segment were calculated according to the international standard (F2259-10(2012)), and 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 exert the catalytic activity of mannouronic acid 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.

[0048] Table 1

[0049] Example 3

[0050] This embodiment provides a type of kelp. MC5E The analysis of gene expression patterns under high temperature stress mainly includes the following steps:

[0051] 1. High-temperature stress treatment experiment;

[0052] 2. Kelp RNA extraction and cDNA synthesis;

[0053] 3. qRT-PCR analysis.

[0054] The specific steps are as follows:

[0055] 1. High-temperature stress treatment experiment: Kelp larvae with a length of 20-30 cm were collected from aquaculture rafts in Rongcheng City, Shandong Province. After rinsing the kelp samples, they were subjected to high-temperature stress at 25℃ and light intensity of 35±5 μmol / m². 2 The samples were cultured at a photoperiod of 12:12 (L:D) with a photoperiod of 12:12. Samples were taken at 0, 1, 3, 6, 12 and 24 h. All samples were wiped dry, flash-frozen in liquid nitrogen and stored at -80℃.

[0056] 2. Kelp RNA Extraction and cDNA Synthesis: Kelp RNA was extracted using the method described in Example 1 of this invention. Evo M-MLV The Mix Kit with gDNA Clean for qPCR synthesizes first-strand cDNA for qRT-PCR detection.

[0057] 3. qRT-PCR analysis: Actin (F: 5'-GACGGGTAAGGAAGAACGG-3'; R: 5'-GGGACAACCAAAACAAGGGCAGGAT-3') were used as internal reference genes to design the present invention. MC5E Gene-specific primers qMC5E-F (5'-CATCGAGGACAGCGAGATGG-3') and qMC5E-R (5'-ACCCCGAAGTTCATGTGGTG-3') were used for quantitative experiments.

[0058] 4. Prepare the reaction system according to the instructions for 2×SYBR Green qPCR Mix (With ROX). The reaction program is as follows: pre-denaturation at 94°C for 3 min; then 40 cycles: denaturation at 94°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 30 s. Each template is repeated four times. -△△Ct The relative quantitative results were calculated using this method. The results show that with the extension of the high-temperature treatment time, the [result] described in this invention [is / is / etc.]. MC5E Gene transcription levels showed an increasing trend. At 3 h, 6 h, 12 h, and 24 h of high-temperature treatment, the gene transcription levels were 1.10, 8.70, 7.58, and 34.02 times higher than those in the control group, respectively. Figure 5 This indicates that the gene is involved in the response of kelp to high-temperature stress, as described in this invention. MC5E Genes can be used in the screening or identification of heat-resistant germplasm.

[0059] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A mannouronic acid C5-isomerase gene, characterized in that: The nucleic acid sequence of this gene is shown in SEQ ID NO.1 in the sequence listing.

2. The protein encoded by the kelp mannuronic acid C5-isomerase gene according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.2 in the sequence listing.

3. The use of the encoded protein of claim 2 in catalyzing the conversion of mannuronic acid to guluronic acid.

4. The application of the protein-encoding preparation according to claim 2 in increasing the content of guluronic acid in alginate and reducing the M / G ratio of alginate.

5. A mannulate C5-isomerase gene expression vector, characterized in that: An expression vector containing the mannuronic acid C5-isomerase gene as described in claim 1 as the target gene.

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