Application of a protein enhancing the immunity of laver and its preparation method

Abstract

The present invention first proposes an application of a protein for enhancing the immunity of laver and a preparation method thereof. The Pyropia yezoensis is soaked in sterilized seawater containing 10 μM Pp07886 protein for 1 day, which can induce the immunity of laver, improve the immune level of laver, and reduce the harm caused by red rot disease. Pp07886 can enhance the expression of innate immune genes of laver and effectively reduce the sensitivity of laver to Pythium porphyrae.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an application of a laver immunity-enhancing protein and a preparation method thereof. Background Art

[0002] Pythium rapae is the primary pathogen of laver red rot. Pythium marinum, P. chondricola, and Alternaria sp. can also cause laver red rot symptoms. Cultivated laver (Pyrrophyta yezoensis, Pyrrophyta haitanensis, and Pyrrophyta lanuginosa) in China, Japan, and South Korea are regularly affected by this disease, resulting in yield reductions of 30-50%, with yields exceeding 60% in some areas. Red rot has also been reported in wild laver in the United States, New Zealand, and the Philippines. The frequency and severity of red rot are closely related to seawater temperature, salinity, water flow, cultivation density, management, and geographic location. Pythium porphyra has a wide tolerance range to temperature and salinity, allowing it to survive in marine sediments, irrigation water, soil, wetlands, wrinkled carrageenan (Chondrus crispus), rice (Oryza sativus), and soybean (Glycine max). Under laboratory conditions, it causes root rot and oospore formation in carrots (Daucus carota subsp. sativus), cucumbers (Cucumis sativus L.), barley (Hordeum vulgare var. hexasichon), lettuce (Lactuca sativa L.), cabbage (Brassica rapa subsp. pekinensis), onions (Allium cepa L.), pumpkins (Cucurbita moschata Duch.), radishes (Raphnus sativus L.), rice (Oryza sativa), tomatoes (Solanum lycopersicum L.), and wheat (Triticum sp.), all of which cause crop death, slow growth, or symbiosis. The wider range of existence and stronger survival ability enable Pythium spp. to survive the summer when there is no laver cultivation in the sea area through various means (saprophytic, parasitic and oospore) and become the source of pathogens in the next cultivation season.

[0003] Currently, the pathogen is primarily controlled through physical and chemical methods such as drying out, refrigeration, and pickling, but these methods are only effective in the early stages of the disease. Pythium porphyrae's wide host range and strong resistance to stress allow it to survive in a variety of environments. Once thick-walled oospores form, the disease becomes difficult to control. Studies have reported that Pythium porphyrae oospores can survive for six months at -20°C and for 2-3 hours in organic acids with a pH of 2-3. Therefore, the development of new control methods is imperative.

[0004] The cell wall is a physical barrier against microbial attack in algae and plants. Plant pathogens can secrete a series of cell wall-degrading enzymes (CWDEs) to break down the cell wall, which not only enables the pathogen to invade host tissues but also provides nutrients for the pathogen. Cell-degrading enzymes include glycoside hydrolases, polysaccharide lyases, and esterases. Among them, glycoside hydrolases are a class of carbohydrate-active enzymes widely distributed in pathogenic fungi and oomycetes, which can catalyze the hydrolysis of glycosidic bonds into glycosides, polysaccharides, and glycoconjugates. The CAzY database classifies glycoside hydrolases into 173 families based on predicted structural and sequence similarities (http: / / www.cazy.org / ). In fungi and oomycetes, some glycoside hydrolase families play important roles in pathogen invasion and pathogenesis. Moreover, some glycoside hydrolases can be recognized by the host as pathogen-associated molecular patterns (PAMPs) to trigger immune responses. For example, in the oomycete pathogen Phytophthora sojae, the GH12 family protein XEG1 is not only an essential virulence factor during infection but also a PAMP factor that can induce host immune responses and cell death in soybean and Solanaceae plants; the GH7 family protein PsGH7a has also been shown to promote the invasion of Phytophthora and trigger hypersensitive cell death in different plant species. Comparative genomic analysis shows that the genomes of many oomycete plant pathogens contain abundant glycoside hydrolases. However, the functions of most glycoside hydrolases are still unclear, especially in oomycetes.

[0005] Porphyra lacks cell-based adaptive immune responses, but it has constitutive innate immune responses, which are mostly inductive or triggerable. This immune response can not only save metabolic costs but also reduce the risk of pathogen adaptation. Porphyra C-type lectin can recognize oligosaccharides released by the degradation of the Porphyra cell wall by pathogens as PAMPs to further activate the immune response. Porphyra responds to pathogen infection by inducing reactive oxygen species, expressing enzymes that degrade the pathogen cell wall, upregulating signal transduction and secondary metabolism levels, and initiating programmed cell death. Expressing and purifying a Porphyra pathogen PAMP factor to induce Porphyra immunity can improve the immune level of Porphyra and reduce the damage caused by red rot disease. Summary of the Invention

[0006] The object of the present invention is to first propose an application of a protein for improving the immunity of Porphyra and its preparation method.

[0007] The present invention is achieved by the following technical solutions:

[0008] To achieve the above object, the present invention provides an application of a protein for improving the immunity of Porphyra. Immerse Porphyra yezoensis in sterilized seawater containing 10 μM Pp07886 protein for 1 day, which can induce the immunity of Porphyra, improve the immune level of Porphyra, and reduce the damage caused by red rot disease.

[0009] Preferably, the above-mentioned pp07886 protein has a sequence reference SeqNo.1.

[0010] Preferably, the above soaking conditions are 15 °C, light intensity 62.5 μmol photons / m 2 s, and the photoperiod L:D = 12:12.

[0011] The present invention also provides a method for preparing a protein that enhances the immunity of laver as follows:

[0012] Use Primer 5.0 software to design primers Seq No.2 and Seq No.3 containing restriction enzyme sites BamH1 and Xho1, and use PrimerSTAR Max DNA Polymerase to perform full-length amplification of the ORF of the Pp07886 gene in the cDNA of NBRC NO.33253. After digesting the Pp07886 sequence with the predicted signal peptide sequence removed, it is ligated to the pGEX4T-1 plasmid digested with BamH1 and Xho1 double enzymes respectively, and then sequenced for verification.

[0013] Escherichia coli BL 21(Beijing Biomed Gene Technology Co., Ltd., Beijing, China) was used as the expression host and cultured in LB liquid medium (containing 100 μg / mL Amp) at 37 °C and 250 rpm for 10 h. IPTG was added to a final concentration of 100 μM, and the culture was transferred to 20 °C and induced at 100 rpm for 24 h. The medium was removed by centrifugation at 4 °C and 10,000 g for 5 min. The precipitate was resuspended in Breaking buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.5) and sonicated on ice for 10 min. After centrifugation at 4 °C and 10,000 g for 5 min, the supernatant and precipitate were collected. The precipitate was dissolved in Wash I (20 mM Tris-HCl, 0.5 M NaCl, 8 M Urea, pH 7.5), and then the supernatant and precipitate were subjected to SDS-PAGE. The target band was cut out and sent to the State Key Laboratory of Genetic Engineering in Shanghai Fuda University for mass spectrometry determination. In order to obtain the active and pure Pp07886 fusion protein, the GST Fusion Protein Purification Kit (GenScript USA, Inc., Nanjing, China) was used for protein purification, and the protein concentration was determined using the Total Protein Assay Kit (with standard: BCA method) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) Pp07886 can increase the expression of innate immune genes in Porphyra;

[0016] (2) Pp07886 can effectively reduce the sensitivity of Porphyra to Pythium porphyrae;

[0017] (3) The present invention first proposes an application method of Pp07886;

[0018] (4) The present invention first proposes an effective preparation method of Pp07886. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the SDS-PAGE electrophoresis diagram of Pp07886 of the present invention; among them, lane 1 is the cell lysate supernatant of the positive clone; lane 2 is the purified Pp07886 protein;

[0020] Figure 2 Mass spectrometry analysis of the Pp07886 protein of the present invention; the gray font part represents the mass spectrometry determination sequence consistent with the expectation.

[0021] Figure 3 qRT-PCR analysis of the transcriptional levels of innate immune genes in Porphyra of the present invention; wherein a is the infection stage. b is the co-culture stage with rPp07886. The mean and standard deviation are for 3 biological replicates. The significant differences between groups were detected by Duncan's analysis in SPSS 20.0 (P<0.05) and represented by letters. CL: C-lectin; NOX: NADPH oxidase; POX: catalase; F3GT: flavonol 3-O-glucosyltransferase; F3H: flavonoid 3ˊ-hydroxylase; UCE2: ubiquitin-conjugating enzyme E2; ULE3: ubiquitin ligase E3.

[0022] Figure 4 qRT-PCR analysis of the transcriptional changes of innate immune genes during the co-culture of Porphyra with GST protein of the present invention; the mean and standard deviation are for 3 biological replicates. The significant differences between groups were detected by Duncan's analysis in SPSS 20.0 (P<0.05) and represented by letters; wherein CL: C-lectin; NOX: NADPH oxidase; POX: catalase; F3GT: flavonol 3-O-glucosyltransferase; F3H: flavonoid 3ˊ-hydroxylase; UCE2: ubiquitin-conjugating enzyme E2; ULE3: ubiquitin ligase E3.

[0023] Figure 5 Effect of the recombinant Pp07886 protein of the present invention on the infection of Pythium porphyrae on Porphyra; wherein a is the observation 7 days after Pythium porphyrae infects Porphyra. b is the observation 7 days after Pythium porphyrae infects Porphyra pretreated with GST for 1 day. c is the observation 7 days after Pythium porphyrae infects Porphyra pretreated with the recombinant Pp07886 protein for 1 day.

[0024] Figure 6 Effect of the recombinant Pp07886 protein of the present invention on the infection of Pythium porphyrae on Porphyra; wherein d is the disease rot rate of Pythium porphyrae infection, the disease rot rate (%) = lesion area / leaf area * 100%. e is the cumulative infection rate of Pythium porphyrae infection, the cumulative infection rate (%) = the number of Porphyra with lesions / total number of Porphyra * 100%. f is the average lesion area of Pythium porphyrae infecting Porphyra, the average lesion area = lesion area / number of lesions. g is the average number of lesions of Pythium porphyrae infecting Porphyra, the average number of lesions = total number of lesions / total number of leaves. Detailed implementation manners

[0025] The content of the present invention will be described more specifically below in conjunction with embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification or change made to the present invention falls within the protection scope of the present invention; and the methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0026] Example 1:

[0027] Pythium porphyrae and porphyra cultivation

[0028] Pythium porphyrae NBRC 33253 was used in this experiment. The marginal mycelium of Pythium porphyrae growing on a semi-seawater corn medium was inoculated into a semi-seawater L-sodium glutamate glucose medium (SGG) and cultured at 24 °C and 100 rpm in the dark. The culture solution was changed every 7 days to obtain mycelia.

[0029] The pure line RZ of Pyropia yezoensis was provided by the Laboratory of Marine Biology Genetics and Breeding, Ocean University of China and used for the infection experiment. RZ was aerated and cultured at 10 °C, light intensity 62.5 μmol photons / m 2 s, light cycle L:D = 12:12. PES was added to the boiled seawater

[381] as the culture solution and changed once every 3 days.

[0030] RNA extraction and cDNA synthesis

[0031] Total RNA of Pythium porphyrae was extracted using the Fungal RNA Kit (Omega Bio-Tek, Inc., Norcross, Georgia, USA) according to the instructions; total RNA of Pyropia yezoensis was extracted using the Plant RNA Kit (Omega Bio-Tek, Inc., Norcross, Georgia, USA). The concentration and purity were measured using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). 1 μg of total RNA was used for cDNA synthesis using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the instructions.

[0032] Cloning, expression and purification of the Pp07886 gene

[0033] Primer 5.0 software was used to design primers containing restriction enzyme sites BamH1 and Xho1. The ORF of the Pp07886 gene in the cDNA of NBRC NO.33253 was amplified in full length using PrimerSTAR Max DNA Polymerase. After digestion of the Pp07886 sequence with the predicted signal peptide sequence removed, it was ligated to the pGEX 4T-1 plasmid digested with BamH1 and Xho1 double enzymes and then sequenced for verification. The primer sequences were Seq No.2 and Seq No.3.

[0034] Escherichia coli BL 21 (Beijing Biomed Gene Technology Co.,Ltd.,Beijing,China) was used as the expression host and cultured in LB liquid medium (containing 100 μg / mL Amp) at 37 °C and 250 rpm for 10 h. The inducer IPTG was added to a final concentration of 100 μM, and the culture was transferred to 20 °C and induced at 100 rpm for 24 h. The culture medium was removed by centrifugation at 4 °C and 10000 g for 5 min. The precipitate was resuspended in Breaking buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.5) and then ultrasonically disrupted on ice for 10 min. After centrifugation at 4 °C and 10000 g for 5 min, the supernatant and precipitate were collected. The precipitate was dissolved in Wash I (20 mM Tris-HCl, 0.5 M NaCl, 8 M Urea, pH 7.5), and then the supernatant and precipitate were subjected to SDS-PAGE. The target band was cut out and sent to the State Key Laboratory of Genetic Engineering in Shanghai Fuda University for mass spectrometry determination. To obtain the active and pure Pp07886 fusion protein, the GST Fusion Protein Purification Kit (GenScript USA, Inc., Nanjing, China) was used for protein purification, and the protein concentration was determined using the Total Protein Assay Kit (with standard: BCA method) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0035] As Figure 1 shown, SDS-PAGE analysis showed that the purified recombinant fusion protein presented as a single band with a molecular weight of approximately 79 kDa. As Figure 2 shown, through mass spectrometry analysis, it was found that 80% of the amino acids in the purified protein matched the Pp07886 sequence, indicating that the expressed protein was Pp07886.

[0036] Pythium porphyrae infects porphyra

[0037] Pythium porphyrae hyphae were placed in a conical flask containing a semi-seawater solution with 10 mM CaCl2 at a concentration of 2 g / L and cultured at 15 °C and 100 rpm for 1 h; the hyphae were taken out and placed in a new conical flask with a semi-seawater solution of 10 mM CaCl2 and cultured for 1 h; the culture was repeated 5 h; after the last replacement of the 10 mM CaCl2 semi-seawater solution, it was cultured at 15 °C and 100 rpm for 12 - 14 h, and then the hyphae were placed in a new 10 mM CaCl2 semi-seawater solution and cultured for 1 h under the above conditions to obtain a large number of zoospores at the same developmental stage.

[0038] Thirty pure-line Pyropia yezoensis (length 5.85 ± 1.14 cm, width 1.05 ± 0.20 cm) were selected and placed in a 250 mL conical flask containing 200 mL of Pythium porphyrae NBRC NO. 33253 zoospore suspension (10 5 CFU / mL), and left standing at 15 °C, light intensity 62.5 μmol photons / m 2 s, and a light cycle of L:D = 12:12 for 9 d. Healthy porphyra without zoospore suspension under the same conditions was used as a control, and 3 parallels were set for each group. One porphyra was taken from each bottle at 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, and 9 d after infection for quantitative analysis of immune genes.

[0039] Analysis of the expression of innate immune genes during the infection of Pythium porphyrae on porphyra

[0040] Using the β-Actin gene of Pyropia yezoensis as an internal reference, the expression patterns of innate immune-related genes (C-lectin, Hsp20, NADPH oxidase, Peroxidase, Flavonol 3-O-glucosyltransferase, β-glucosidase, Ubiquitin-conjugating enzyme E2 and Ubiquitin ligase E3) in Pyropia yezoensis during the infection process were quantitatively detected. Axygen Strip Tubes (Coring Life Sciences (Wujiang) Co., Ltd., Jiangsu, China) were used for qRT-PCR analysis. The reaction system was 20 μL, including: 10 μL Green Realtime PCR Master Mix (2×) (TOYOBO Co., Ltd., Biotech support Department, Osaka, Japan), 1 μL forward primer, 1 μL reverse primer, 7 μL DNase / RNase-free water (TIANGEN Biotech Co., Ltd., Beijing, China), and 1 μL template. qRT-PCR was performed using a CFX Connect thermal cycler (Bio-Rad Laboratories, Linc., California, USA). The reaction program was as follows: pre-denaturation at 95 °C for 1 min, followed by denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s, and fluorescence reading, for 40 cycles.

[0041] Effect of recombinant Pp07886 protein on innate immune genes of Porphyra

[0042] For detecting the effect of Pp07886 protein on the innate immunity of Porphyra, 20 pure-line Porphyra yezoensis (length 5.31 ± 0.92 cm, width 1.06 ± 0.15 cm) were placed in a 250 mL conical flask containing 100 mL of sterilized seawater (containing 10 μM Pp07886 protein), and left standing for 7 d at 15 °C, light intensity 62.5 μmol photons / m 2 s, and light cycle L:D = 12:12. GST protein with a final concentration of 10 μM added under the same conditions was used as a control. Three parallels were set for each group. Every day, 1 Porphyra was taken from each bottle, snap-frozen in liquid nitrogen, and RNA was extracted using a Plant RNA Kit (Omega Bio-Tek, Inc., Norcross, Georgia, USA). cDNA synthesis and quantification of innate immune genes were as described above.

[0043] As Figure 3 and Figure 4 shown, during the infection of Porphyra thallus by Pythium porphyrae, all the selected immune genes were strongly induced at the mid-stage of infection (day 5) ( Figure 3 a). In the co-culture experiment, it was found that compared with the co-culture of Porphyra and GST protein, during the co-culture of Porphyra thallus and rPp07886, the transcriptional levels of 5 genes (CL, Hsp20, F3GT, F3H, and UCE2) reached their peaks on day 1 (23, 55, 702, 20, and 52 times that of uninfected Porphyra respectively), indicating that these genes responded rapidly to rPp07886 ( Figure 3b). Additionally, ULE3 and POX showed a slower response to rPp07886, with the highest transcriptional levels on day 3 (4 times that of the uninfected Porphyra) and day 6 (26 times that of the uninfected Porphyra), respectively ( Figure 3 b). However, compared with the control group, there was no significant difference in the transcriptional level of NOX ( Figure 4 ). Therefore, Pp07886 can activate the expression of some innate immune genes in Porphyra. The rapid response of the transcription of Porphyra immune genes co-incubated with rPp07886 may be due to Pp07886 being recognized as PAMPs by Porphyra and strongly stimulating the innate immune system.

[0044] Effect of recombinant Pp07886 protein on the infection of Pythium porphyrae

[0045] For detecting the effect of Pp07886 protein on the infection of Pythium porphyrae, 20 pure-line Porphyra yezoensis (length 5.26 ± 0.83 cm, width 1.03 ± 0.12 cm) were placed in a 250 mL conical flask containing 100 mL of sterilized seawater (containing 10 μM Pp07886 protein), and after standing for 1 d at 15 °C, light intensity 62.5 μmol photons / m 2 s, and light cycle L:D = 12:12, zoospores of Pythium porphyrae were added to a final concentration of 10 5 CFU / mL. GST protein at 10 μM and the group without adding any protein were used as controls. After 7 d of infection, the disease rot rate, cumulative infection rate, number of disease spots, and disease spot area were counted.

[0046] As Figure 5 and Figure 6 shown, after treating Porphyra with recombinant Pp07886 protein for 1 d and then conducting the infection experiment, it was found that the disease rot rate, average number of disease spots, and average disease spot area in the treatment group were significantly lower than those in the control group, about 75% of the control group, but there was no obvious difference in the disease rot rate. This indicates that recombinant Pp07886 can effectively improve the resistance of Porphyra to Pythium porphyrae, but it cannot make it completely immune to infection.

[0047] In summary, our results demonstrate that Pp07886 not only enhances the expression of innate immune genes in Porphyra but also effectively reduces its susceptibility to Pythium porphyrae. We hypothesize that during the initial stages of Pythium porphyrae infection, Pp07886 is recognized by the C-lectin pattern recognition receptor in Porphyra and activates PTI. POX expression is induced to continuously produce reactive oxygen species, inhibiting pathogen infection. Simultaneously, F3GT and F3H are activated, resisting pathogen invasion by regulating the production of hesperidin and other flavonoids. Hsp20 is induced to repair damage, which is important for the stability and accumulation of antipathogen proteins and the coordination of the entire defense signaling cascade. Furthermore, the induction of two ubiquitin proteins, UCE2 and ULE3, is closely linked to the regulation of plant immunity. The specific regulatory and activation mechanisms require further investigation.

[0048] The specific implementation manner described above is a preferred implementation manner of the present invention, and is not intended to limit the specific implementation scope of the present invention. The scope of the present invention includes but is not limited to this specific implementation manner. All equivalent changes made in accordance with the present invention are within the protection scope of the present invention.

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

Claims

1. Use of a protein that enhances the immunity of laver, characterized in that, Soak Porphyra yezoensis in sterilized seawater containing 10 μM Pp07886 protein for 1 day, which can induce the immunity of Porphyra yezoensis, improve the immune level of Porphyra yezoensis, and reduce the harm caused by red rot disease; the sequence of the Pp07886 protein is as shown in Seq No.1; the pathogenic bacterium of the red rot disease is Pythium porphyrae.

2. The application of the protein for enhancing the immunity of laver according to claim 1, wherein The soaking conditions are 15 °C, light intensity 62.5 μmol photons / m2s, and photoperiod L:D = 12:12.

Images