Application of Japanese larch CHS1 gene or encoding protein thereof in improving pine wood nematode resistance of pine trees
By overexpressing the CHS1 gene in Japanese larch, its resistance to pine wood nematode was enhanced, solving the problem of pine trees being easily infected and achieving effective control of pine wood nematode, with significant economic and ecological benefits.
Patent Information
- Application Number
- CN202511968596.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-17
AI Technical Summary
Existing technologies are insufficient to effectively improve pine trees' resistance to pine wilt disease, leading to pine trees being susceptible to infection and dying rapidly, resulting in economic and ecological losses.
By overexpressing the CHS1 gene of Japanese larch or enhancing the activity of its encoded protein, the CHS1 gene was introduced into pine trees using a recombinant plant expression vector, thereby enhancing their resistance to pine wood nematode.
It significantly improves the resistance of pine trees to pine wilt disease, reduces nematode reproduction and mortality, protects the health of pine trees, and has important economic and ecological value.
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Figure CN121674465A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to CHS1 New uses for genes, particularly involving Japanese larch CHS1 The use of genes or their encoded proteins in improving pine resistance to pine wood nematode belongs to CHS1 New applications of genes or their encoded proteins. Background Technology
[0002] Pine wilt disease, also known as pine wilt, is a devastating disease of pine trees, often referred to as the "cancer" of pine. It first appeared in China in 1982, introduced from Japan. Once a pine tree is infected, it dies within about six weeks. If timely control measures are not implemented, large areas of pine forests can be destroyed within 3-5 years. Therefore, it is crucial to strengthen the prevention and control of pine wilt disease (Yu Lanchen. Research on the harm and integrated control strategies of pine wilt disease [J]. Agricultural Development and Equipment, 2020(7):233). Currently, biological control of pine wilt mainly involves releasing natural enemies of the vector insects, such as the pine sawyer beetle, in the forest to control the density of these insects.
[0003] Japanese larch is a high-quality, fast-growing timber species with a long history of introduction to China, possessing significant economic and ecological value. Pine wilt nematode infection causes stress to Japanese larch, with the accumulation of large amounts of ROS leading to cell necrosis, decreased water transport capacity, and further disruption of water conduction, resulting in more extensive cell damage and causing seedling needle wilting (Wang Jianguo et al. Effects of pine wilt nematode infection on physiological indicators of Japanese larch. Journal of Southwest Forestry University. Vol. 43, No. 6). Therefore, improving the resistance of Japanese larch to pine wilt nematode will have significant economic and ecological value. Summary of the Invention
[0004] The main objective of this invention is to provide a gene or its encoded protein that can be used to improve the resistance of pine trees to pine wood nematode.
[0005] This invention discovers that Japanese larch CHS1 Overexpression of the gene in embryogenic callus of Japanese larch can significantly improve the resistance of embryogenic callus to pine wood nematode, thus completing this invention.
[0006] This invention mainly provides Japanese larch CHS1 The use of the gene or its encoded protein (CHS1 protein) in enhancing pine resistance to pine wood nematode; As a possible implementation scheme, it can be achieved by using... CHS1 Genes are overexpressed or enhanced in pine trees CHS1 The activity of the gene-encoded protein increases the resistance of pine trees to pine wood nematode.
[0007] In another aspect, the present invention provides a method for improving the resistance of pine trees to pine wilt disease, comprising: overexpressing in pine trees... CHS1 Genes, making CHS1 Increased gene expression may enhance the function or activity of the encoded protein; for example, by... CHS1 Genes are operably linked to expression regulatory elements to obtain recombinant plant expression vectors that express the gene in plants; these recombinant plant expression vectors are then transformed into pine trees to... CHS1 The gene was overexpressed in pine trees, and the resulting transgenic pine trees showed enhanced resistance to pine wood nematode.
[0008] The invention described CHS1 The nucleotide sequence of the gene's CDS is selected from any of the nucleotide sequences described in (a)-(e) below: (a) The polynucleotide sequence shown in SEQ ID No. 2;
[0009] (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; MAAGMMKDLEAFRKAQRADGPATILAIGTATPANAVDQSTYPDYYFKITNSEHMTELKEKFQRMCDKSAIKKRYMYLTEEILKENPKVCEYMAPSLDARQDMVVVEVPRLGKEAATKAIKEWGQPKSKITHVIFCTTSGVDMPGADYQLTKLLGLRPSVKRVMMYQQGCFAGGTVLRVAKDLAENNRGARVLVVCSEVT AVTFRGPSDTHLDSMVGQALFGDGAAALIVGADPIPQVEKPCFELLWTAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLVDAFQQFGISDWNELFWIAHPGGPAILDQVEAKLNLDPKKLGATRKVLSEYGNMSSACVHFILDEMRKSSQEKGCSTTGEGLNVGVLFGFGPGLTVETVVLKSVPLQ (SEQ ID No.1).
[0010] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions; (d) A polynucleotide sequence that has at least 95% or more identity with any of the polynucleotide sequences shown in (a)-(c) and has the function of enhancing the resistance of pine wood nematode.
[0011] The percentage of sequence identity described in this invention can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.
[0012] In addition, those skilled in the art can optimize the nucleotides shown in SEQ ID No. 2 to enhance their expression efficiency in plants.
[0013] The present invention can also produce mutants by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to the 5' end and / or 3' end of the amino acid sequence of the protein encoded by its gene.
[0014] The amino acid sequence of the CHS1 protein described in this invention is selected from any of the following amino acid sequences (a)-(d): (a) The amino acid sequence shown in SEQ ID No. 1; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1, which still has the function or activity of improving the resistance of pine trees to pine wood nematode; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID No. 1, which still has the function or activity of improving the resistance of pine trees to pine wood nematode; (d) A protein with more than 95% identity to the amino acid sequence shown in SEQ ID No. 1, which still has the function or activity of improving the resistance of pine trees to pine wood nematode.
[0015] The protein described in this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.
[0016] Another aspect of the present invention is to further provide a product containing the aforementioned... CHS1 The gene expression cassette, recombinant vector, or recombinant host cell; preferably, the expression cassette, recombinant vector, or recombinant host cell is a recombinant eukaryotic expression cassette, recombinant eukaryotic expression vector, or recombinant plant cell; more preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or recombinant plant expression vector.
[0017] For reference, the present invention provides CHS1 Gene recombinant expression vectors for plants, including: Will CHS1A recombinant plant expression vector is obtained by operatively linking a gene to an expression regulatory element. This recombinant plant expression vector may consist of a 5′ untranslated region, the nucleotides shown in SEQ ID No. 2, and a 3′ untranslated region. The 5′ untranslated region may include a promoter sequence, an enhancer sequence, and / or a translational enhancement sequence. The promoter may be a constitutive promoter, an inducible promoter, or a tissue- or organ-specific promoter. The 3′ untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. A suitable terminator sequence can be obtained from the Ti-plasmid of *Agrobacterium tumefaciens*, such as the terminator regions of octopine synthase and carmine synthase.
[0018] The recombinant plant expression vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicides. Furthermore, the marker genes may also include phenotypic markers, such as β-galactosidase and fluorescent proteins.
[0019] The transformation scheme and the scheme for introducing the polynucleotide or polypeptide into the plant can vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment. In specific embodiments, various transient transformation methods can be used to transform alfalfa. MtMET1 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).
[0020] The pine trees mentioned in this invention include larch, Chinese pine, podocarpus, or Scots pine; wherein, the larch is preferably Japanese larch.
[0021] Definitions of terms involved in this invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0022] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.
[0023] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
[0024] In this invention, "rigorous hybridization conditions" refers to conditions of low ionic strength and high temperature known in the field. Typically, under rigorous conditions, the detectability of the probe hybridizing with its target sequence is significantly higher than that with other sequences (e.g., at least twice the background level). Rigorous hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the rigor of hybridization or washing conditions. Detailed guidance on nucleic acid hybridization can be found in relevant literature (Tijssen, ...). Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, (Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are typically chosen to be below the melting point (T0) of the specific sequence at a specified ionic strength pH. m Approximately 5-10℃. m The temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence under equilibrium conditions (at specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is present in excess, therefore at T...). m(Under equilibrium conditions, 50% of the probe is occupied). Strict conditions may include: a salt concentration of less than approximately 1.0 M sodium ions at pH 7.0 to 8.3, typically approximately 0.01 to 1.0 M sodium ions (or other salts), and a temperature of at least approximately 30 °C for short probes (including, but not limited to, 10 to 50 nucleotides) and at least approximately 60 °C for long probes (including, but not limited to, greater than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal may be at least twice the background hybridization, and, where appropriate, 10 times the background hybridization. Exemplary strict hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42 °C; or 5×SSC, 1% SDS, incubated at 65 °C, washed in 0.2×SSC and washed in 0.1% SDS at 65 °C. The washing can be performed for 5, 15, 30, 60, 120 minutes or longer.
[0025] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.
[0026] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, an engineered selection marker for expression in plant cells, and the heterologous DNA sequence to be transcribed.
[0027] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.
[0028] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells. Attached Figure Description
[0029] Figure 1 For the transfer CHS1 Morphological changes of Japanese larch callus inoculated with aseptic pine wood nematode. Detailed Implementation
[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.
[0031] Experimental Example 1: Japanese Larch CHS1 Tests on resistance of transgenic larch embryogenic callus to pine wood nematode Based on the analysis of the chalcone synthase gene family of Japanese larch and the metabolomics data of Korean pine infection with pine wilt nematode previously obtained in the laboratory, genes with relatively high expression levels were selected. CHS1 Genes are used for genetic transformation experiments.
[0032] 1. Experimental Methods Gene cloning and vector construction: Total RNA was extracted from embryogenic callus of Japanese larch using an RNA extraction kit, and obtained by RT-PCR. CHS1 The cDNA of the gene was extracted and amplified by PCR. The PCR product and 1 μg of Cam35S-GFP plasmid were digested and purified by enzymes. The digested gene and vector were ligated using full-length gold T4 ligase, transformed into competent E. coli cells, and positive clones were screened using LB plates containing Kan (50 μg / mL).
[0033] Table 1. Japanese Larch CHS1 Gene amplification primer sequences
[0034] Agrobacterium-mediated genetic transformation: In the recipient material preparation stage, larch callus tissue was placed in BM medium (1.43 / L BM + 0.5 mg / L 6-BA + 0.05 mg / L KT + 0.5 mg / L NAA + 1000 mg / L glutamine + 500 mg / L acid-hydrolyzed casein + 25 g / L sucrose + 4 g / L agar + pH 5.8) and incubated in the dark at 23°C for 5 days. Then, it was transferred to an Erlenmeyer flask containing 150 mL of liquid BM medium and incubated in the dark at 23°C with shaking at 100 rpm for 7 days. 50 μL of Escherichia coli culture was added to 5 mL of LB liquid medium (containing 15 μL of 50 mg / mL Kan) and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were then extracted. Plasmids were transformed into Agrobacterium using a liquid nitrogen flash-freezing method. The bacterial culture was then evenly spread onto LB solid medium (containing 50 µg / mL Kan, 1 ml per 1 L; 100 µg / mL Rif, 1 ml per 1 L) and incubated upside down at 28°C for 48 h. Single colonies were then inoculated into liquid LB medium (containing Kan and Rif) and cultured at 28°C with agitation at 200 rpm until the bacterial concentration reached OD500. 600nmCentrifuge at 0.6-0.8, 4000 rpm, 4℃ for 10 min, discard the supernatant, and suspend the recombinant Agrobacterium in infection medium (liquid BM+AS, 1L with 200ul AS), adjusting the OD600nm to 0.1. After the embryogenic cells have completed culture, dry them using a vacuum filtration sterilization device, place them in the Agrobacterium infection medium for 10 min, dry them again, and transfer them to solid BM+AS medium. Incubate at 23℃ in the dark for 48 h. Then, pour the embryogenic cells into a vacuum filtration sterilization device, wash them four times with sterile water (3 min each time), and then wash them four times with 500 mg / L Cef aqueous solution (8 min each time). Collect the embryogenic cells and transfer them to solid BM+Cef (500 mg / L) medium lined with sterile filter paper, incubate at 23℃ in the dark for 7 days. After co-culture, surviving embryogenic cells were collected and transferred to solid BM+cef (500 mg / L)+hyg (5 mg / L) medium, and cultured in the dark at 25°C for 20 days. This process was repeated three times to obtain larch embryogenic callus.
[0035] Using the obtained larch embryogenic callus as experimental material, each 0.5g of embryogenic callus was inoculated with 50μL of sterile pine wood nematodes (containing a total of 50 nematodes) as the transgenic experimental group. The uninfected (untransformed) group was used as the control group. CHS1 Embryogenic callus (genotype) was inoculated with 50 μL of sterile pine wood nematodes per 0.5 g of embryogenic callus (totaling 50 nematodes) as the wild-type experimental group. Five days after inoculation, the morphology of the larch embryogenic callus and the resistance of the pine wood nematodes were observed. The nematode reproduction rate, including the number of live and dead nematodes, was recorded, and the mortality rate was calculated using the following formula: Mortality rate = Number of dead insects / Total number of insects.
[0036] Criteria for determining nematode death: The nematode is considered dead when it is immobile, its body is bent in a "C" or "J" shape, and it does not respond to mechanical stimulation.
[0037] 2. Experimental Results According to the experimental results in Tables 2 and 3, for the wild-type experimental group, the number of nematodes reproduced on day 5 after inoculation of 0.5g callus tissue with 50μL of sterile pine wood nematodes was 1072, of which 10 nematodes died, resulting in a nematode mortality rate of 0.93%. For the transgenic experimental group, due to better resistance than the wild type, only 314 nematodes reproduced on day 5 after inoculation of 0.5g callus tissue with 50μL of sterile pine wood nematodes. The nematode reproduction rate was significantly lower than that of the wild type, and 41 of the 314 nematodes died, resulting in a mortality rate of 13.06%.
[0038] Table 2. Pine wood nematode reproduction rate (nematodes / 0.5g) after inoculating 0.5g of callus with sterile pine wood nematodes.
[0039] Table 3 Mortality rate of pine wood nematodes after inoculation of 0.5g callus with aseptic pine wood nematodes
[0040] according to Figure 1 The experimental results showed that, after inoculation with pine wood nematodes, the larch embryogenic callus tissue exhibited overall browning and tissue collapse compared to callus tissue inoculated with sterile water. In contrast, the WT group showed a significantly different outcome. CHS1 The relatively mild damage to the callus tissue indicates that the genome is overexpressed in larch. CHS1 Genes can enhance the resistance of larch trees to pine wilt nematodes.
Claims
1. Japanese larch CHS1 Use of a gene or its encoded protein in increasing resistance of a pine tree to pine wood nematode.
2. Use according to claim 1, characterized in that, The genes were overexpressed or upregulated in pine trees CHS1 The genes were overexpressed or upregulated in pine trees CHS1 The activity of the encoded proteins of the genes increased the resistance of the pine trees to pine wood nematodes.
3. Use according to claim 1, characterized in that, The CHS1 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) - (d) below: (a) a polynucleotide sequence as set forth in SEQ ID No. 2; (b) a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No. 1; (c) a polynucleotide sequence capable of hybridizing to the polynucleotide sequence as set forth in (a) or (b) under stringent hybridization conditions; (d) a polynucleotide sequence having at least 95% or more identity to the polynucleotide sequence as set forth in any one of (a) to (c), which polynucleotide sequence has a function of enhancing the resistance of Bursaphlenchus xylophilus.
4. Use according to claim 1, characterized in that, The amino acid sequence of the protein is selected from any one of the following (a) to (d): (a) an amino acid sequence as set forth in SEQ ID No. 1; (b) a protein variant obtained by deletion or substitution of one or more amino acid residues in the amino acid sequence as set forth in SEQ ID No. 1, which protein variant still has a function or activity of enhancing the resistance of pine trees to Bursaphlenchus xylophilus; (c) a protein variant obtained by insertion of one or more amino acid residues in the amino acid sequence as set forth in SEQ ID No. 1, which protein variant still has a function or activity of enhancing the resistance of pine trees to Bursaphlenchus xylophilus; (d) a protein having 95% or more identity to the amino acid sequence as set forth in SEQ ID No. 1, which protein still has a function or activity of enhancing the resistance of pine trees to Bursaphlenchus xylophilus.
5. Use according to claim 1, characterized in that, The plant includes larch, oil pine, podocarpus or scots pine.
6. Use according to claim 5, characterized in that, The larch is Japanese larch.
7. A method of increasing resistance of a pine tree to Bursaphelenchus xylophilus comprising: overexpressing in pine CHS1 genes, which increase the expression amount of CHS1 genes, which increase the expression amount of or enhance the function or activity of the protein encoded by The resulting transgenic pine tree has enhanced resistance to Bursaphlenchus xylophilus.
8. The method of claim 7, wherein, Will CHS1 Genes are operably linked to expression regulatory elements to obtain recombinant plant expression vectors that express the gene in plants; these recombinant plant expression vectors are then transformed into pine trees to... CHS1 The gene was overexpressed in pine trees.
9. The method of claim 7, wherein, The CHS1 The nucleotide sequence of the CDS of the gene is selected from any one of the nucleotide sequences described in (a) - (d) below: (a) a polynucleotide sequence as set forth in SEQ ID No. 2; (b) a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No. 1; (c) a polynucleotide sequence capable of hybridizing to the polynucleotide sequence as set forth in (a) or (b) under stringent hybridization conditions; (d) a polynucleotide sequence having at least 95% or more identity to the polynucleotide sequence as set forth in any one of (a) to (c), which polynucleotide sequence has a function of enhancing the resistance of Bursaphlenchus xylophilus.
10. The method of claim 7, wherein, The pine tree includes larch, oil pine, podocarpus or scots pine; preferably, the larch is Japanese larch.