Application of KMFP1967 gene and protein thereof in preparation of algal phosphorus stress detection reagent

Abstract

The invention discloses an application of a KMFP1967 gene and a protein thereof in preparation of a reagent for detecting algae phosphorus stress. According to the application disclosed by the invention, through multi-omics analysis of transcriptomics, translational omics and proteomics, the key effect of the KMFP1967 protein in the phosphorus absorption and metabolism process of the karella mikimotoi is deeply verified. The significant up-regulation of the protein not only reveals the core molecule regulation mechanism of the Karenia mikimotoi for dealing with phosphorus stress, but also indicates the unique function of the Karenia mikimotoi in protein folding and endoplasmic reticulum processing, provides a brand new molecular target for red tide prevention and control, lays a technical development foundation for red tide treatment, and has a wide application prospect. And meanwhile, valuable theoretical basis and practical support are provided for related industrial application.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a KMFP1967 gene and a protein thereof in preparing a reagent for detecting algae phosphorus stress. Background Art

[0002] Harmful Algal Blooms (HABs) are a major threat to the global marine ecosystem and coastal economy, causing billions of dollars in economic losses to aquaculture and environmental protection, and posing potential risks to human health. Among the many algae that cause red tides, Karenia mikimotoi has caused serious damage to marine life and the balance of the ecosystem due to its widespread distribution and the hemolytic toxins and fish toxins released during outbreaks.

[0003] Although the red tide problem has attracted much attention, the genome structure of Karenia mikimotoi is still complex and has not yet been fully analyzed. This limitation has led to current research focusing mainly on the transcriptomics level. However, most transcriptome data are only sequence information, which makes it difficult to directly reveal the expression dynamics of functional proteins and the metabolic mechanisms they participate in. Therefore, in-depth analysis of the phosphorus absorption and metabolic mechanisms of Karenia mikimotoi and the exploration of molecular targets that may play a key role in the formation of red tides are scientific and technological challenges that need to be overcome urgently. Summary of the invention

[0004] Phosphorus is an important nutritional factor for the growth of red tide algae and has a significant impact on its growth rate and toxicity. In response to this key point, this study used differential proteomics technology to compare the protein expression patterns of Karenia mikimoto under normal and phosphorus stress conditions, and successfully identified a key differential protein molecule that was significantly upregulated in the protein synthesis and regulatory pathway. Further verification at the transcription and translation levels using qRT-PCR technology confirmed that the protein has a significant expression regulatory effect in phosphorus metabolism. This finding suggests that the protein may play an important role in the process of phosphorus absorption and metabolism, and may be an important molecular regulatory factor in the formation of red tides.

[0005] This invention clarifies for the first time the importance of this key protein in the phosphorus metabolism of Karenia mikimotoi, filling the gap in the research field of functional proteins of red tide algae. This discovery not only provides a new perspective for understanding the formation mechanism of red tides, but also lays a theoretical foundation and application direction for the development of innovative solutions for red tide management. By targeting this protein molecule, it is expected to achieve precise intervention and prevention and control of red tide outbreaks.

[0006] Karenia mikimotoi has always been a major challenge in red tide research and intervention due to its complex genome structure and low consistency between mRNA and protein expression. Due to the complexity of the genome and the lack of mature genetic intervention methods, existing technologies are difficult to deeply analyze its metabolism and toxicity mechanisms at the DNA level, resulting in slow progress in related research. At the same time, existing research mainly focuses on transcriptomics data analysis, but this type of technology cannot fully reflect the expression and role of key functional proteins in phosphorus metabolism and red tide formation.

[0007] In proteomics research, the protein extraction process of Karenia mikimotoi was seriously interfered by polysaccharides and polyphenols. These substances not only reduced the purity of protein extraction, but also had a negative impact on subsequent analysis, greatly limiting the accuracy and reliability of the data. These technical bottlenecks have become the core issues restricting the development of red tide research and control technology.

[0008] Based on the above technical difficulties, the present invention proposes an innovative research method, combining differential proteomics and transcriptomics technology to overcome the problem of polysaccharide and polyphenol interference in the traditional protein extraction process. Through the optimized protein extraction and purification process, as well as differential protein screening under phosphorus stress conditions, the present invention successfully identified a key differential protein that was significantly upregulated during phosphorus metabolism. Further verification by qRT-PCR technology showed that the protein showed significantly high expression at both the transcription and translation levels, indicating its important role in phosphorus metabolism and red tide formation.

[0009] The innovative features of the present invention include: Identification and functional verification of key proteins: For the first time, the key differential proteins of Karenina mikimoto were systematically screened and verified under phosphorus stress conditions, providing important clues for revealing the mechanism of red tide formation.

[0010] Optimized protein extraction technology: By improving the protein extraction and purification process, the interference problems of polysaccharides and polyphenols were effectively solved, greatly improving the accuracy and reliability of proteomics data.

[0011] Integration and innovation of research methods: By integrating differential proteomics, transcriptomics and translationomics technologies, functional analysis from RNA to protein is achieved, providing a new research paradigm for the study of red tide algae.

[0012] This invention provides a new target for the study of phosphorus metabolism mechanisms and lays a solid foundation for the development of precise intervention strategies for red tide outbreaks. By targeting this key differential protein, it is expected to provide a new solution for red tide prevention and control.

[0013] The present invention is achieved through the following technical solutions: Application of KMFP1967 gene and its protein in the preparation of reagents for detecting algal phosphorus stress.

[0014] Preferably, the sequence of the KMFP1967 gene is as shown in SEQ ID NO.1.

[0015] Preferably, the algae are dinoflagellates.

[0016] Preferably, the dinoflagellate is Gymnodinium.

[0017] Preferably, the algae is Karenia mikimotoi.

[0018] Preferably, the method comprises the following steps: (1) Obtain the expression level of the KMFP1967 gene under normal conditions, which is defined as the standard value; (2) Obtain the expression level of the KMFP1967 gene under the test state, which is defined as the test value; (3) When the test value is greater than the standard value, the algae are under phosphorus stress.

[0019] Preferably, step (3) is specifically as follows: when the test value is greater than or equal to 1.5 times the standard, the algae are under phosphorus stress; and the expression amount in step (1) or step (2) is the transcription amount or translation amount of the gene.

[0020] The invention also provides application of KMFP1967 gene and protein thereof as algae phosphorus stress markers.

[0021] Preferably, the sequence of the KMFP1967 gene is as shown in SEQ ID NO.1; and the algae is Karenia mikimotoi.

[0022] The present invention has but is not limited to the following beneficial effects: This invention deeply verifies the key role of KMFP1967 protein in phosphorus absorption and metabolism of Karenia mikimotoi through multi-omics analysis of transcriptomics, translationomics and proteomics. The significant upregulation of this protein not only reveals the core molecular regulatory mechanism of Karenia mikimotoi in response to phosphorus stress, but also shows its unique function in protein folding and endoplasmic reticulum processing, providing a new molecular target for red tide prevention and control, laying a foundation for the development of technology for red tide control, and providing valuable theoretical basis and practical support for related industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to clearly demonstrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0024] Figure 1 Changes in cell division and alkaline phosphatase activity (APA) during the culture of Karenia mikimotoi over culture time (days). (A) Changes in alkaline phosphatase activity over culture time. (B) Changes in cell division over culture time.

[0025] Figure 2 Differential proteome analysis of Karenia mikimoto under phosphorus stress (A) Venn diagram of proteins identified by ASTRAL and LUMOS of Karenia mikimoto; (B) Volcano plot of the intersection of proteins identified by ASTRAL and LUMOS of Karenia mikimoto; (C) KEGG pathway diagram of the intersection of proteins identified by ASTRAL and LUMOS of Karenia mikimoto; (D) GO analysis diagram of the intersection of proteins identified by ASTRAL and LUMOS of Karenia mikimoto; (E) Up-regulated proteins (KMFP1967) in endoplasmic reticulum folding and processing at the intersection of proteins identified by ASTRAL and LUMOS of Karenia mikimoto.

[0026] Fig. 3 qRT-PCR results of KMFP1967 gene transcriptome level in normal group and phosphorus stress group of Karenia mikimotoi. ** indicates P ≤ 0.01, blue is the expression level of KMFP1967 in normal group, and red is the expression level of KMFP1967 in phosphorus stress group.

[0027] Specific implementation methods The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments belong to part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results all belong to the protection scope of the present invention.

[0028] Example 1 The present invention relates to Karenia mikimotoi ( Karenia mikimotoi ) Under phosphorus stress conditions, a multi-omics combined analysis method based on proteomics, transcriptomics and translationomics is aimed at identifying and verifying key proteins related to phosphorus metabolism. Through the integrated analysis of multi-omics data, the present invention successfully discovered specific molecules related to phosphorus absorption, metabolism and regulation in Karenia mikimotoi, especially the protein KMFP1967 that was significantly upregulated under phosphorus stress. Its function in protein folding and endoplasmic reticulum processing makes it a core molecule for phosphorus metabolism regulation, providing a theoretical basis and experimental data support for revealing the molecular mechanism of Karenia mikimotoi's adaptation to low-phosphorus environments.

[0029] Before describing the technical solution of the present invention, the terms used in this document are first defined to ensure the consistency and accuracy of the terms: The term "qRT-PCR" refers to: quantitative reverse transcription polymerase chain reaction, which is a molecular biology technique used to quantify the expression levels of specific RNA.

[0030] The term “KEGG” refers to: Kyoto Encyclopedia of Genes and Genomes, which is a database including genomes, chemical substances, and system functions, widely used in bioinformatics research, especially gene and metabolic pathway analysis.

[0031] The term "Spectronaut" refers to: a software for mass spectrometry data analysis, mainly used for processing and analyzing mass spectrometry data, especially widely used in quantitative proteomics.

[0032] The term "DAPI" refers to: a fluorescent dye commonly used for cell staining that can bind to DNA and emit blue fluorescence under ultraviolet light and is commonly used for cell nucleus staining.

[0033] The term "SYBR" refers to: A common fluorescent dye commonly used in real-time quantitative PCR (qPCR) experiments that binds to DNA and emits green light in fluorescent detection systems.

[0034] The term "Tris-HCl" refers to: a commonly used buffer component, usually used in DNA and RNA extraction and electrophoresis experiments to maintain a stable pH value.

[0035] The term "DTT" refers to: dithiothreitol, which is commonly used to reduce disulfide bonds in proteins and is often used as a reducing agent during protein extraction and folding.

[0036] The term "cDNA" refers to complementary DNA, which refers to DNA synthesized by the process of reverse transcription and is commonly used to study gene expression and construct gene expression libraries.

[0037] The term "PMSF" refers to: phenylmethylsulfonyl fluoride, a common protease inhibitor widely used in cell lysates to prevent protein degradation.

[0038] The term "SDS" refers to sodium dodecyl sulfate, a detergent commonly used in protein electrophoresis that disrupts the three-dimensional structure of proteins and provides negative charges that cause them to migrate according to molecular size during electrophoresis.

[0039] The term "TCA" means: trichloroacetic acid.

[0040] The term "DIA" refers to: Data Independent Acquisition. A mass spectrometry data acquisition mode that improves the quantitative accuracy and coverage of mass spectrometry by simultaneously acquiring all ion signals compared to traditional data-dependent acquisition (DDA). It is commonly used in high-throughput proteomics analysis.

[0041] To achieve the above objectives, the present invention established a culture system for Karenia mikimoto under phosphorus stress conditions, systematically carried out transcriptomics, translationomics and proteomics research, deeply analyzed the biological response of Karenia mikimoto under phosphorus stress, and combined with qRT-PCR to verify the role of the key differential protein KMFP1967 in protein synthesis and regulation.

[0042] 1. Cultivation of K. mikimotoi and phosphorus stress treatment: First, the algae were cultured under different phosphorus concentrations to simulate phosphorus stress in the natural environment. The growth state and phosphorus metabolism changes of the algae under different stress conditions were determined by adjusting the phosphorus concentration.

[0043] 2. RNA extraction of transcriptome and translatome and transcriptome third-generation sequencing Based on the first step, the second aspect of the present invention extracts RNA from the transcriptome and translation group to ensure high-quality sample extraction for subsequent deep sequencing and data analysis. The transcriptome is sequenced using the PacBio platform for third-generation sequencing, and a protein reference database of Karenia mikimotoi is constructed.

[0044] 3. Construction and analysis of differential proteomes of K. mikimoto under phosphorus stress In the third aspect of the present invention, by adopting efficient protein extraction and separation technology, the differential proteome of Karenia mikimotoi under normal group and phosphorus stress group was successfully constructed. The protein reference library of the second aspect was combined to perform Spectronaut library search. The library search results were subjected to KEGG differential protein functional enrichment analysis, and a highly expressed differential protein KMFP1967 mainly focused on protein folding under phosphorus stress was found.

[0045] 4. qRT-PCR validation of transcriptome and translationome based on proteome results In the fourth aspect of the present invention, based on the analysis results of the differential proteome, qRT-PCR experiments were further performed at the transcriptome and translationome levels to verify the high expression of KMFP1967. By comparing and analyzing the transcriptome data, the consistency of the key proteins upregulated under phosphorus stress at the transcriptional and translational levels was confirmed, providing strong support for subsequent functional studies and target verification.

[0046] To achieve the purpose, the present invention adopts the following technical solutions: Establishment of a culture system for K. mikimotoi under phosphorus stress

[0047] Karenia mikimotoi (* Karenia mikimotoi*, strain number: HK-5) was provided by the Algae Collection Center of the Harmful Algae and Marine Biology Research Center of Jinan University. In this study, algae strains were cultured in the normal phosphorus concentration group (Control group) and the phosphorus stress group (-P group), and the specific culture conditions were as follows: In the normal group, the algae strains were cultured in f / 2-Si medium, the inorganic phosphate (NaH2PO4) concentration in the medium was 36 μmol L⁻¹, the light intensity was 100 μmol photons m⁻² s⁻¹, and the photoperiod was set to 12 h light: 12 h dark. The phosphorus stress group was cultured for two generations through continuous subculture, and the inorganic phosphate in the culture medium was gradually reduced to about 1 μmol L⁻¹ to adapt the algae cells to the low phosphorus environment. All experimental samples were collected during the exponential growth phase (about 8-10 days). During the experiment, DAPI and SYBR staining combined with confocal microscopy observation confirmed that the algae cell culture state was normal and there was no bacterial contamination. Finally, about 900 mL of algae liquid was collected from each group for proteomics, third-generation sequencing, transcriptomics, and translationomics analysis. As shown in Figure 1, the growth curves of the normal group and the phosphorus stress group showed that the algae cells grew well, which was in line with the experimental expectations.

[0048] RNA extraction of transcriptome and translatome and transcriptome third-generation sequencing Given that there is still a lack of complete genome information for Karenia mikimotoi, this project combines the third-generation sequencing technology with the advantage of ultra-long read length to identify the mRNA sequences being translated by Karenia mikimotoi for the construction of a protein reference library. The ribosome nascent chain complex (RNC) separation method is as follows. The Karenia mikimotoi algae liquid was first centrifuged (5000g, 5 minutes, 4°C), the precipitate was collected and centrifuged again (5000g, 5 minutes, 4°C), and the obtained precipitate was ground in liquid nitrogen. After sufficient grinding, 2 ml of pre-cooled cell lysis buffer (200 mM Tris-HCl, 35 mM MgCl2, 200mM KCl, 10mMDTT, 10 μg / ul actinomycin, 1% Triton X-100) was used for lysis for 15 minutes. The cell lysate was then clarified by centrifugation at 12,000 × g for 15 min at 4°C, and the supernatant was transferred to a 12.5 ml sucrose cushion (30% sucrose, 202 mM Tris-HCl, 35 mM MgCl2, 200 mM KCl, 10 mM DTT, 10 μg / ul cycloheximide). RNCs were purified by ultracentrifugation at 380,000 × g, 4°C for 4.5 h in a 50.2Ti rotor (Optima XPN-100, Beckman, USA). Total RNC-RNA was finally isolated from K. mikimotoi using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).

[0049] As for the total RNA of Karenia mikimotoi, we extracted the total RNA of Karenia mikimotoi using RNeasy Plus Mini Kit and removed the genomic DNA using RNase-free DNase I. Then, the integrity of RNA was tested by Agilent 2100 Bioanalyzer and agarose gel electrophoresis. The purity and concentration of RNA samples were evaluated by Nanodrop microspectrophotometer and Qubit 2.0 fluorometer, respectively. We selected RNA samples with 260 / 280 ratio and 260 / 230 ratio greater than or equal to 1.8 and RIN value greater than or equal to 5 for the construction of Pacbio sequencing library.

[0050] During the library construction process, 5 μg of total RNA (equal amounts of each RNA were mixed) was first used for SMRT library construction. According to the Isoform sequencing protocol, the mRNA was converted into cDNA through a reverse transcription process using the Clontech SMARTer PCR cDNA Synthesis Kit. Next, the PCR reaction was optimized to determine the optimal number of large-scale PCR amplification cycles. Using the selected number of amplification cycles and Phusion DNA polymerase, a large amount of double-stranded cDNA was generated.

[0051] Afterwards, the Blue Pippin™ Size-Selection System was used to screen out cDNA molecules longer than 5 kb and mix them with equal amounts of cDNA that had not been size-selected. After another round of large-scale PCR amplification of this mixture, the resulting cDNA product was used to prepare the SMRTbell template library. Finally, the Karenia mikimotoi sample was sequenced and data analyzed on the PacBioBioscience Sequel platform. A total of 97.14 Gb of raw data was obtained by three-generation sequencing. After de-redundant processing, 21,922 de-redundant representative sequences were obtained, providing comprehensive transcriptome information for subsequent proteomics analysis.

[0052] Construction of a proteome reference library of Kaplania mirmannii Before the differential proteome analysis, there was no protein reference library for Karenia mikimotoi because there was no genomic information. Therefore, the following method was used to construct the differential proteome. First, the transcriptome data obtained from the third-generation sequencing of Karenia mikimotoi was first translated into six frames: the Biopython library of python was used to read the transcriptome gene sequence file in FASTA format and extract the sequence data. Then, each mRNA sequence was translated into six frames, including three forward translation frames and three reverse complementary translation frames. The specific steps include: 1) Forward translation: translation starts from the 1st, 2nd, and 3rd nucleotide positions of each sequence respectively; 2) Reverse complementary translation: the reverse complementary sequence of each sequence is translated into the same three frames. After the six-frame translation, the protein sequence is cut. The specific steps are: first, the sequence is split according to the stop codon, and then the promoter ('M') in each fragment is searched. For each promoter found, the sequence fragment from 'M' to the terminator is extracted. Each fragment is saved as an independent sequence. After protein translation, we used cd-hit software to remove redundancy from the sequences, with the following parameters: sequences with similarity greater than 0.95 were grouped into one category; short sequences with similarity greater than 0.99 were grouped into one category. Finally, the final Karenia mikimotoi protein reference library was obtained.

[0053] Construction and analysis of differential proteomes under phosphorus stress in K. mikimotoi In terms of differential proteomics, 3 × 100 ml of the phosphorus bacteria solution of Michaelis mikrophore were centrifuged at 5000 g for 5 minutes in the normal group and the phosphorus stress group, most of the supernatant was removed by pouring, the precipitate was resuspended with the remaining supernatant and centrifuged again, and the algae precipitate was transferred to a grinding tube. The grinding tube was placed in a pre-cooled grinder and ground (70 Hz, 5 minutes). After thorough grinding, SDS containing 1% PMSF was added for ultrasonic lysis (60 power, 5 seconds on, 5 seconds off, 10 minutes, below 10°C), the supernatant was taken and dried as much as possible, and then the protein was purified by TCA-acetone precipitation method (add 6 volumes of pre-cooled acetone solution containing 10% TCA, incubate at -20°C overnight, centrifuge at 8000 g for 10 min at 4°C on the second day, aspirate the supernatant, rinse with 1 ml pre-cooled acetone, centrifuge at 8000 g for 10 min, take the supernatant and retain the precipitate) to remove polysaccharide and polyphenol impurities. According to the instrument experimental instructions, the purified protein sample was enzymatically hydrolyzed and desalted using a PreON automatic enzymolyzer (Preomics, Germany), and finally the Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ and Orbitrap™ Astral™ instruments were used for DIA mode on-machine identification. Using the above protein reference library and computer files, 5167 proteins were finally identified, of which 3050 were classified as intersection high confidence proteins ( Figure 2 A).

[0054] Differential protein analysis of 3050 proteins classified as high confidence in the intersection showed that a total of 710 significantly differentially expressed proteins were identified in the phosphorus stress group, of which 328 proteins showed a fold change of more than 1.5 times under phosphorus stress ( Figure 2 B). Under phosphorus stress conditions, the protein expression of Karenia mikimotoi changed significantly. Further GO and KEGG enrichment analysis showed that these proteins were closely related to metabolic reprogramming, regulation of photosynthesis and protein folding regulation ( Figure 2 C and D). In addition, we screened out a protein KMFP1967 (FC=1.77; P-Value<0.01; MW=64321.06) that was significantly upregulated in protein folding regulation from the differentially expressed proteins with a difference of more than 1.5 times. The function of this protein is closely related to protein folding and endoplasmic reticulum processing (see Figure 2 E).

[0055] Example 2 qRT-PCR validation at the transcriptome and translationome levels based on proteome results

[0056] Based on the results of differentially expressed proteins, the present invention intends to Figure 3 A) and the translation team ( Figure 3 B) This protein KMFP1967 was verified at the level. In order to verify the reliability of the results, this example used a sample different from that of Example 1. Specifically, this Example 2 referred to the method of Implementation 1 to culture the algae again, and used the transcriptome and translation group RNA obtained from the above method. According to the manufacturer's instructions, the first-stop cDNA was synthesized using the TransScript cDNA Synthesis Kit (YEASEN, Shanghai, China). According to the manufacturer's instructions, quantitative PCR was performed on the Roche real-time PCR system using the Heieff UNICON qPCR SYBR Green premix (Vazyme, Nanjing, China). The following standard thermal curve was used for all quantitative PCRs: 95°C for 3 minutes; 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 20 seconds. GPADH was used as the internal reference. The primers used for expression analysis are listed in Table 1. The final result analysis showed (see Figure 3 ), the expression of KMFP1967 in the transcriptome and translationome was consistent with its changing trend in the differential proteome.

[0057]

[0058] Example 3 Results and Discussion

[0059] The present invention solves the research problem of the adaptation mechanism of K. mikimoto under phosphorus stress by combining proteomics, transcriptomics and translationomics analysis methods, overcomes many bottlenecks in the prior art, and has the following technical advantages: 1. Breakthrough in the identification and verification of key proteins in phosphorus metabolism in a complex genomic context

[0060] The genome of Karenia mikimotoi is complex and its mRNA and protein expressions have low consistency, which makes it difficult for existing technologies to conduct in-depth research on its metabolic mechanism and toxicity mechanism from the DNA level. The present invention successfully identified and verified the key protein KMFP1967 related to phosphorus metabolism through a multi-omics combined analysis method, solving the problem of the difficulty of genomic research in traditional research. This method can more accurately reveal the biological response of Karenia mikimotoi under phosphorus deficiency conditions, providing important data support and theoretical basis for subsequent phosphorus metabolism intervention research.

[0061] 2. Overcome interference during protein extraction and ensure data accuracy and reliability In existing proteomics research, when extracting proteins from Karenia mikimotoi, polysaccharides and polyphenols are often interfered with, which affects protein purification and subsequent analysis, and reduces the accuracy and reliability of data. The present invention proposes an optimized protein extraction method, successfully solves these interference problems, greatly improves the accuracy and reliability of data, and provides a more reliable basis for subsequent proteomics analysis.

[0062] 3. Multi-omics combined analysis fills the research gap of existing technologies and provides a more comprehensive analysis of molecular mechanisms Current research techniques mainly focus on transcriptomic data analysis, which is difficult to effectively reflect the functional expression of key proteins in phosphorus metabolism and red tide formation. The present invention provides a more comprehensive and in-depth analysis of molecular mechanisms by combining proteomics, transcriptomics and translationomics. This innovative method makes up for the limitations of the single existing technology and can more accurately reveal the metabolic mechanism and adaptive response of Karenia mikimotoi under phosphorus stress.

[0063] 4. Other microalgae species can be used instead of Karenia mikimotoi The present invention focuses on Karenia mikimotoi ( Karenia mikimotoi ) under phosphorus stress conditions to identify and verify the key protein KMFP1967. However, other microalgae species with similar phosphorus metabolism characteristics, such as red tide-related dinoflagellate species (such as Alexandrium, Gymnodinium, etc.), can also be used as alternatives for similar experiments. In the case of changing the microalgae species, as long as the key protein with similar function to KMFP1967 can be identified and its role in phosphorus metabolism can be verified, the research purpose of the present invention can be achieved. Therefore, other microalgae species can be used as research objects, but the core technology still revolves around the identification and verification of key proteins similar to KMFP1967.

[0064] 5. Scope of protection for specific protein molecules The core of the present invention is to identify and verify the key protein KMFP1967 that is closely related to phosphorus metabolism in Karenia mikimotoi, and propose the functional role of these proteins in phosphorus metabolism. Although the present invention currently focuses on KMFP1967, the identification method and application of this molecule and its functional regulatory mechanism can also be extended to other proteins with homology or functional similarity. For example, proteins related to phosphorus metabolism in other microalgae species (such as molecules with functional domains or biological effects similar to KMFP1967) can be identified and verified by the method of the present invention, thereby forming patent protection for these protein molecules. This alternative scheme still takes KMFP1967 as the technical core while expanding the scope of protection.

[0065] 6. Alternative methods for analyzing phosphorus absorption and metabolic pathways The present invention uses proteomics technology to deeply analyze KMFP1967, a key protein in the process of phosphorus absorption and metabolism, and combines functional annotations to infer its metabolic pathway. As an alternative, metabolomics methods can be used to directly measure the changes in metabolites during phosphorus metabolism, thereby indirectly inferring enzymes related to the function of KMFP1967 and their regulatory mechanisms. This alternative method infers changes in the activity of key proteins by detecting changes in metabolite concentrations, which is particularly suitable for situations where gene or protein expression data are relatively scarce. Although the analysis methods are different, the ultimate goal is still to confirm key molecules similar to KMFP1967 and their core role in phosphorus metabolism.

Claims

1. Application of KMFP1967 gene and its protein in the preparation of reagents for detecting algal phosphorus stress.

2. The use according to claim 1, characterized in that The sequence of the KMFP1967 gene is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The algae are dinoflagellates.

4. The use according to claim 3, characterized in that The dinoflagellate is a naked dinoflagellate.

5. The application according to claim 1, characterized in that: The algae is Karenia mikimotoi.

6. The application as claimed in claim 1, characterized in that The steps include: (1) Obtain the expression level of the KMFP1967 gene under normal conditions, which is defined as the standard value; (2) Obtain the expression level of the KMFP1967 gene under the test state, which is defined as the test value; (3) When the test value is greater than the standard value, the algae are under phosphorus stress.

7. The use according to claim 6, characterized in that: Step (3) is specifically as follows: when the test value is greater than or equal to 1.5 times the standard, the algae are under phosphorus stress; the expression amount in step (1) or step (2) is the transcription amount or translation amount of the gene.

8. Application of KMFP1967 gene and its protein as algal phosphorus stress markers.

9. The use according to claim 8, characterized in that The sequence of the KMFP1967 gene is shown in SEQ ID NO.1; the algae is Karenia mikimotoi.

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