Gold potted plant'sharp leaf' mitogen-activated protein kinase armapk20, coding gene and application

Abstract

The present application relates to a kind of Anoectochilus roxburghii 'Jianye' mitogen-activated protein kinase ArMAPK20, coding gene and application, the above-mentioned protein kinase ArMAPK20 includes the polypeptide with the amino acid sequence as shown in SEQ ID NO.2.This application also provides the nucleotide sequence SEQ ID NO.1 for encoding the above-mentioned protein.This application provides a theoretical basis for improving the ability of Anoectochilus roxburghii to resist stem rot by using genetic engineering technology, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering application technology, and relates to an important mitogen-activated protein kinase in the disease resistance process of Anoectochilus roxburghii, specifically involving the mitogen-activated protein kinase ArMAPK20 of the 'tip leaf' of Anoectochilus roxburghii, its encoding gene and application. Background Technology

[0002] *Anoectochilus roxburghii* (Wall.) Lindl., belonging to the genus *Anoectochilus* in the family Orchidaceae, is a rare perennial medicinal plant in my country. The whole plant is used medicinally, possessing properties such as clearing heat and detoxifying, calming the liver and tonifying the kidneys, earning it the title of "King of Herbs." However, *Anoectochilus roxburghii* is susceptible to pathogen infection during cultivation, severely interfering with and damaging its normal growth. Stem rot is a common disease in the artificial cultivation of *Anoectochilus roxburghii*, characterized by rapid onset, wide impact, and high damage rate; in severe cases, the infection rate can reach 90%, reducing yield by 70%–80%. Currently, the control of stem rot in *Anoectochilus roxburghii* mainly relies on chemical control, supplemented by physical and biological control methods. However, the intrinsic defense mechanisms of *Anoectochilus roxburghii* against pathogen invasion have not yet been systematically elucidated.

[0003] Mitogen-activated protein kinase (MAPK) is a highly conserved core component of signal transduction in eukaryotes. In plants, it converts external signals such as pathogen infection into intracellular molecular responses through a three-tiered kinase cascade of "MAPKKK-MAPKK-MAPK," serving as a crucial molecular bridge connecting pattern recognition receptors and downstream disease resistance responses. MAPK plays an irreplaceable core role in plant disease defense, acting as a key hub for immune signal amplification and precise regulation. However, there are currently no research reports on the involvement of MAPK proteins in the resistance of *Anoectochilus roxburghii* to stem rot. Summary of the Invention

[0004] To fill the gaps in the cloning, expression pattern, and protein information of the ArMAPK20 gene in *Anoectochilus roxburghii*, this invention provides the nucleotide and amino acid sequences of the mitogen-activated protein kinase ArMAPK20, its transient infection resistance phenotype, and its subcellular protein localization. This provides a theoretical basis for future use of genetic engineering techniques to regulate ArMAPK20 gene expression and improve the resistance of *Anoectochilus roxburghii* to stem rot.

[0005] On the one hand, the present invention provides ArMAPK20, a mitogen-activated protein kinase of Anoectochilus roxburghii with the function of promoting resistance to stem rot, wherein the protein kinase ArMAPK20 comprises a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO.2; or a protein having the characteristics of ArMAPK20 of Anoectochilus roxburghii with one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.2.

[0006] On the other hand, the present invention provides a coding gene encoding the above-mentioned *Anoectochilus roxburghii* 'tip leaf' mitogen-activated protein kinase ArMAPK20, wherein the nucleotide sequence of the coding gene is specifically: (a) the base sequence is as shown in positions 1 to 1182 of SEQ ID NO.1; or (b) a sequence having at least 70% homology with the nucleotides shown in positions 1 to 1182 of SEQ ID NO.1.

[0007] In this invention, "isolated DNA" and "purified DNA" refer to DNA or fragments that have been separated from sequences flanking them in their natural state, and also to DNA or fragments that have been separated from components that accompany nucleic acids in their natural state, and from proteins that accompany them in the cell.

[0008] In this invention, the gene encoding ArMAPK20, the mitogen-activated protein kinase of *Anoectochilus roxburghii* 'tip leaf', refers to the nucleotide sequence encoding a polypeptide with *Anoectochilus roxburghii* 'tip leaf' mitogen-activated protein kinase activity, such as the nucleotide sequence from positions 1 to 1182 of SEQ ID NO.1 and its degenerate sequence. This degenerate sequence refers to a sequence generated when one or more codons in positions 1 to 1182 of SEQ ID NO.1 are replaced by degenerate codons encoding the same amino acid. Due to codon degeneracy, a degenerate sequence with less than 70% homology to the nucleotide sequence from positions 1 to 1182 of SEQ ID NO.1 can also encode the sequence shown in SEQ ID NO.2. The aforementioned encoding gene can also be a nucleotide sequence with at least 70% homology to the nucleotide sequence shown in SEQ ID NO.1.

[0009] The aforementioned coding gene can also refer to variants that encode the same function as the natural *Anoectochilus roxburghii* mitogen-activated protein kinase ArMAPK20, with the sequence shown in SEQ ID NO.1. These variants include (but are not limited to): deletions, insertions, and / or substitutions typically of 1 to 120 nucleotides, and additions of up to 90 nucleotides at the 5' and / or 3' ends.

[0010] In this invention, the expression pattern of the ArMAPK20 gene product of *Anoectochilus roxburghii* 'pointed leaf' can be analyzed using real-time quantitative PCR, that is, the presence and quantity of the mRNA transcript of the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' can be analyzed in cells.

[0011] Furthermore, based on the nucleotide and amino acid sequences of the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' according to the present invention, homologous genes or homologous proteins related to the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' can be screened based on nucleic acid homology or expressed protein homology.

[0012] The full-length nucleotide sequence or fragment thereof related to the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in this invention, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0013] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0014] In addition, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0015] Besides being produced by recombinant methods, fragments of the protein of the present invention can also be produced by solid-phase technology through direct peptide synthesis. The individual fragments of the protein of the present invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

[0016] The present invention also provides a recombinant expression vector containing the encoding gene of ArMAPK20, the mitogen-activated protein kinase of *Anoectochilus roxburghii* 'tip leaf'; the recombinant expression vector is pCAMBIA1300-ArMAPK20.

[0017] The present invention also provides a recombinant bacterium, which is obtained by transforming the above-mentioned recombinant expression vector into a host cell; the host cell is Agrobacterium GV3101.

[0018] The present invention also provides the application of the above-mentioned gene encoding ArMAPK20, a mitogen-activated protein kinase in *Anoectochilus roxburghii* 'tip leaf', in improving the resistance of *Anoectochilus roxburghii* to stem rot.

[0019] Beneficial Effects: As a rare and precious traditional Chinese medicine, *Anoectochilus roxburghii* (Golden Thread Orchid) is in high demand in the market. Stem rot is a common disease in the artificial cultivation of *Anoectochilus roxburghii*, seriously affecting the quality of the medicinal material and industrial benefits. This invention clones for the first time the coding sequence of ArMAPK20, an important regulatory protein kinase in the resistance to stem rot in *Anoectochilus roxburghii*. The expression pattern of the ArMAPK20 gene was analyzed using real-time quantitative PCR, and the subcellular localization of ArMAPK20 was analyzed by transient expression analysis in tobacco leaf epidermal cells. This provides a theoretical basis for future use of genetic engineering technology to regulate the spatiotemporal expression of the ArMAPK20 gene, thereby improving the resistance of *Anoectochilus roxburghii* to stem rot and breeding new disease-resistant varieties, and has great application value. Attached Figure Description

[0020] Figure 1 This document presents the homology comparison results (DNAMAN) of ArMAPK20, a mitogen-activated protein kinase from *Anoectochilus roxburghii* 'tip leaf', and MAPK protein sequences from *Dendrobium* and other sources, as well as a phylogenetic tree analysis of the ArMAPK20 gene with homologous genes from *Arabidopsis thaliana* and other sources. A represents the multiple sequence alignment diagram of ArMAPK20 from *Anoectochilus roxburghii* 'tip leaf' and its homologous proteins; B represents the phylogenetic tree analysis.

[0021] Figure 2 Map showing the localization of ArMAPK20, a mitogen-activated protein kinase from *Anoectochilus roxburghii* 'tip leaf', in tobacco leaf epidermal cells;

[0022] Figure 3 Phenotypic growth of *Anoectochilus roxburghii* 'pointed leaf' after transient infection with the ArMAPK20 gene and subsequent inoculation with *Fusarium oxysporum* (days 0-7);

[0023] Figure 4 This diagram shows the expression changes of the ArMAPK20 gene in *Anoectochilus roxburghii* 'pointed leaf' after infection with *Fusarium oxysporum*. Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0025] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0026] Example 1 Full-length cloning of the ArMAPK20 gene from *Anoectochilus roxburghii* 'pointed leaf'

[0027] Obtaining plant materials: After 7 days of hardening-off and 14 days of normal growth, healthy *Anoectochilus roxburghii* seedlings of the pointed-leaf type were infected with Fusarium oxysporum using a fungal cake method for 7 days to obtain diseased samples; total RNA was extracted from the stems, leaves and other tissues of the diseased *Anoectochilus roxburghii*.

[0028] Total RNA was extracted using the "SteadyPure RNA Extraction Kit" from Hunan Aikerui Biotechnology Co., Ltd. RNA integrity was assessed by gel electrophoresis, and RNA purity and concentration were determined at 280 nm and 260 nm using an ultra-micro spectrophotometer. cDNA was synthesized by reverse transcription using the Evo M-MLV Plus cDNA Synthesis Kit.

[0029] Full-length cloning of the gene: Based on the nucleotide sequence and protein function annotation results provided by the transcriptome sequencing of *Anoectochilus roxburghii*, the full-length ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' was obtained. Primers for the target gene sequence were designed and synthesized by Youkang Biotechnology Co., Ltd. Using *Anoectochilus roxburghii* cDNA 5 days after stem rot as a template, PCR was performed using primers ArMAPK20-F (5'-ATGGACGCCGCGCCCCAGTCGGCGGATG-3') and ArMAPK20-R (5'-CTAGTAGTCTGGGTTGAAGGCAATGCCC-3'), a 1182 bp fragment was amplified. The fragment was recovered and ligated into the pMD18-T vector, and then sent to a sequencing company for sequencing using ArMAPK20-F and ArMAPK20-R as universal primers.

[0030] The sequencing results were combined with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) prediction to obtain the open reading frame of the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf'. The nucleotide sequence of the ArMAPK20 gene of *Anoectochilus roxburghii* 'pointed leaf' was obtained by PCR amplification (SEQ ID NO.1).

[0031] Example 2 Sequence information and homology analysis of the ArMAPK20 gene in *Anoectochilus roxburghii* 'pointed leaf'

[0032] The full-length open reading frame (OPG) sequence of the ArMAPK20 gene from *Anoectochilus roxburghii* 'pointed leaf' in this invention is 1182 bp, and the detailed sequence is shown in SEQ ID NO.1. Based on the OPG sequence, the amino acid sequence of ArMAPK20 from *Anoectochilus roxburghii* 'pointed leaf' was deduced, consisting of 393 amino acids, a molecular weight of 44.79 kDa, and an isoelectric point (pI) of 5.39, and the detailed sequence is shown in SEQ ID NO.2.

[0033] The open reading frame sequence of the ArMAPK20 gene from *Anoectochilus roxburghii* 'pointed leaf' and the amino acid sequence of its encoded protein were analyzed using BLAST in NCBI for nucleotide and protein homology. The results showed that it exhibited extremely high amino acid similarity to genes from *Dendrobium* and other plants. Figure 1 As shown in Figure A. Phylogenetic analysis revealed that the protein kinase ArMAPK20 from *Anoectochilus roxburghii* 'tip leaf' shares high homology with MAPK proteins from other known species such as *Arabidopsis thaliana*. Figure 1 As shown in B.

[0034] Example 3 Construction of recombinant expression vector pCAMBIA1300-ArMAPK20 and subcellular localization analysis of protein kinase ArMAPK20 in tobacco leaves

[0035] Specific primers F (5'-TCGGTACCCGGGGTACCATGGACGCCGCGGC-3') and R (5'-TGCTCACCATGTCGACGTAGTCTGGGTTGAAGGCAATGC-3') were designed at the start and stop codons, respectively, and restriction enzyme sites Kpn I and Sal I were introduced on both sides of the full-length gene sequence. The plasmid containing the target fragment with the restriction sites was double-digested with the pCAMBIA1300 binary transformation vector. The digested vector was recovered and ligated with the ArMAPK20 fragment using T4 ligase at 16℃ for 12-14 h to construct the pCAMBIA1300-ArMAPK20 recombinant expression vector.

[0036] The correct recombinant expression vector was screened by PCR and sequencing. The recombinant plasmid was then transformed into Agrobacterium competent cells GV3101 using the freeze-thaw method to obtain recombinant Agrobacterium GV3101 containing the recombinant expression vector.

[0037] The identified GV3101 strain was inoculated into 5 mL of YEP (containing 50 mg / L Kan) and cultured at 28°C and 180 rpm until OD. 600 The value is approximately 0.6; 1 mL of bacterial culture is added to 25 mL of YEP liquid medium and incubated at 28°C until the OD value reaches 0.6. 600 The OD value was approximately 0.6; 10 mL of bacterial culture was centrifuged at 4500 rpm for 15 min; the bacterial cells were resuspended in MS liquid medium until the OD value reached approximately 0.6. 600Approximately 0.6 mg / L was added, along with AS and MES, and incubated at room temperature for at least 3 hours. This was then injected into tobacco leaves and cultured in the dark for 48 hours. Observation was performed using a laser confocal microscope (GFP excitation wavelength 488 nm, emission wavelength 500-550 nm; DAPI excitation wavelength 405 nm, emission wavelength 420-480 nm). The results showed that the protein kinase ArMAPK20 was localized in the nucleus and cell membrane, such as… Figure 2 As shown.

[0038] Example 4 Growth phenotype of *Anoectochilus roxburghii* 'pointed leaf' ArMAPK20 gene transiently infected with *Anoectochilus roxburghii* followed by inoculation with *Fusarium oxysporum* (days 0-7).

[0039] (1) Pre-shaking Agrobacterium: Select positive Agrobacterium clones containing the pCAMBIA1300-ArMAPK20 recombinant expression vector and add them to 5 mL of YEP liquid medium containing 50 mg / L Kan and 25 mg / L Rif. Incubate at 28°C and 180 rpm for 24 h.

[0040] (2) Propagation of Agrobacterium: The pre-shaken Agrobacterium culture was diluted 1:100 in YEP medium containing the same resistance, and cultured at 28℃ and 200 rpm for 13-16 h until the OD value was reached. 600 The bacterial growth rate was approximately 0.6, and the bacteria were collected at 18°C, 3500 rpm, for 15 minutes.

[0041] (3) Prepare infection buffer (4.43 g / L MS, 10 mM MES, pH 5.6, 200 μM AS). Resuspend the collected Agrobacterium tumefaciens in the infection buffer and adjust the OD. 600 Adjust the pH to 0.6-0.8, and let it stand at room temperature for 2-3 hours to activate it, and the infection solution will be obtained.

[0042] (4) Select tissue culture seedlings or potted seedlings of Anoectochilus roxburghii that have grown for 4-6 weeks and have uniform growth as infection material. Water the plants thoroughly one day before infection to keep the leaves at a high water content, which is conducive to Agrobacterium infection.

[0043] (5) Using a sterile syringe (without the needle), draw up the infection solution and gently press it into the lower epidermis of the *Anoectochilus roxburghii* leaf until the infection solution soaks the entire leaf. Treat 3-4 mature leaves per plant. Plants carrying *Agrobacterium* with an empty vector but not inoculated with the pathogen were used as blank controls (CK), plants carrying *Agrobacterium* with an empty vector and inoculated with the pathogen were used as pathogen controls (Fusarium oxysporum), and plants inoculated with the pathogen after being uniformly sprayed with methyl jasmonic acid (MeJA) (10 μM) exogenously onto the leaves of *Anoectochilus roxburghii* were used as positive controls. MeJA is a derivative of jasmonic acid (JA), an endogenous disease resistance signaling molecule in plants. Exogenous spraying of MeJA can be perceived by plants and activate the JA signaling pathway, triggering the expression of downstream defense genes. It has been proven to enhance the resistance of various plants to pathogens. Therefore, this experiment used MeJA spraying as a positive control to verify the reliability of the experimental system.

[0044] (6) 48 h after transient infection, Fusarium oxysporum pathogen was inoculated at the base of the stem and cultured for another 7 days, and growth phenotype changes were recorded. Symptoms were observed and compared between the transient overexpression group (ArMAPK20), the pathogen control group (Fusarium oxysporum), the blank control group (CK), and the positive control group (MeJA).

[0045] The results are as follows Figure 3 As shown, plants in the pathogen control group developed obvious water-soaked lesions on day 3 after inoculation, the stem base began to turn brown and soften on day 5, and the stem base severely rotted and the plants collapsed and wilted on day 7. Plants in the blank control group showed no symptoms during the 7-day observation period and grew well. Plants in the transient overexpression group showed no obvious symptoms on day 7 after inoculation and maintained upright growth; their disease severity was significantly less than that of the pathogen control group. The positive control group also showed a clear disease resistance phenotype.

[0046] The above results indicate that transient overexpression of the ArMAPK20 gene can delay and alleviate the symptoms of stem rot caused by Fusarium oxysporum infection, suggesting that the ArMAPK20 gene in the 'point leaf' of Anoectochilus roxburghii plays an important positive regulatory role in improving the stem rot resistance of Anoectochilus roxburghii.

[0047] Example 5 The pointed leaves of *Anoectochilus roxburghii* were instantly infected with the ArmAPK20 gene.

[0048] (1) Obtaining materials: After transiently infecting the 'pointed leaf' of *Anoectochilus roxburghii* with the ArMAPK20 gene, *Fusarium oxysporum* mycelium was inoculated 48 h later. Samples were taken on days 0, 1, 2, 3, 4, 5, 6, and 7. The samples were wrapped in aluminum foil, placed in liquid nitrogen, and then stored in an ultra-low temperature freezer at -80℃ for later use.

[0049] (2) RNA extraction, determination of RNA integrity, purity and concentration, and acquisition of cDNA are described in Example 1.

[0050] (3) Design specific primers to perform real-time quantitative PCR analysis of gene expression in Anoectochilus roxburghii stem. Based on the obtained Anoectochilus roxburghii ArMAPK20 gene sequence, specific primers for quantitative analysis of ArMAPK20 gene in Real-time PCR were designed: primer qArMAPK20-F (5'-GGGAAGGGAGCATACGGGAT-3'), primer qArMAPK20-R (5'-GCAGCTTAATCTCCCGCAGT-3'), and the internal reference gene ArActin primers were ArActin-F (5'-GCTAGTGGCCGTACAACTGG-3') and ArActin-R (5'-GCCAGCAAGGTCCAATCGAA-3').

[0051] (4) Standard curves for the target gene and internal reference gene: The standard cDNA solution was serially diluted with ddH2O. Using the diluted cDNA as a template, real-time PCR amplification was performed using specific primers for the target gene and internal reference gene. Melting curves and standard curves were plotted. The melting curves were analyzed to confirm whether the melting curves of the target gene and internal reference gene showed a single peak, thereby verifying the specificity of the primers and the singleness of the PCR amplification products. The appropriate cDNA template dilution factor was determined through the standard curves to provide an accurate quantitative basis for subsequent experiments.

[0052] (5) Real-time fluorescence quantitative analysis of the target gene in the sample to be tested: Using the first strand of the synthesized cDNA as a template, fluorescence quantitative analysis was performed by amplification with specific primers for the target gene and the internal reference gene, respectively. Real-time PCR reaction was performed using a Bio-Rad CFX real-time fluorescence quantitative instrument. The reaction system was 20 µL. The reaction program was: 94℃ pre-denaturation for 20 s, 94℃ for 15 s; 55℃ for 15 s; 72℃ for 15 s; 40 cycles.

[0053] (6) Using 2 -△△Ct Methods for relative quantitative analysis, such as Figure 4 As shown, after infection with *Fusarium oxysporum*, the expression level of the ArMAPK20 gene exhibited a trend of first significantly increasing, then decreasing, and then increasing again. The expression level began to significantly increase on the second day after inoculation, reaching a small peak on the third day, decreasing slightly on the fourth day, and reaching a peak on the fifth day, significantly higher than the uninoculated control. These results indicate that the expression of the ArMAPK20 gene is strongly induced by *Fusarium oxysporum* infection, suggesting its active participation in the immune response of *Anoectochilus roxburghii* to stem rot pathogens.

[0054] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0055] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A type of ArMAPK20 mitogen-activated protein kinase from *Anoectochilus roxburghii* 'pointed leaf', characterized in that, The amino acid sequence of the protein kinase ArMAPK20 is shown in SEQ ID NO.

2.

2. A gene encoding ArMAPK20, the mitogen-activated protein kinase of *Anoectochilus roxburghii* 'tip leaf' as described in claim 1, characterized in that, The encoding gene is a nucleotide sequence as shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, It includes the encoding gene of ArMAPK20, the mitogen-activated protein kinase of *Anoectochilus roxburghii* 'tip leaf' as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is pCAMBIA1300-ArMAPK20.

5. A recombinant bacterium, characterized in that, The recombinant expression vector of claim 4 is obtained by transforming host cells.

6. The recombinant bacteria according to claim 5, characterized in that, The host cell was Agrobacterium GV3101.

7. The application of the gene encoding ArMAPK20, the mitogen-activated protein kinase of *Anoectochilus roxburghii* 'tip leaf' as described in claim 2, in improving the resistance of *Anoectochilus roxburghii* to stem rot.

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