Coffee reverse osmosis protein gene and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a coffee reverse osmosis protein gene and application thereof. The nucleotide sequence of the coffee reverse osmosis protein gene is as shown in SEQ ID NO. 1. The amino acid sequence of the coffee reverse osmosis protein coded by the coffee reverse osmosis protein gene is as shown in SEQ ID NO. 2. The coffee reverse osmosis protein has antibacterial biological activity, can be used for preparing antibacterial products, is applied to prevention and control of crop diseases, and can inhibit growth of Colletotrichum gloeosporioides, sugarcane red rot bacteria and tea leaf blight bacteria.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a coffee reverse osmosis protein gene and its application. Background Technology

[0002] Crop diseases caused by plant pathogenic fungi pose a significant threat to global agricultural production, characterized by diverse infection sites, severe damage, and long duration, significantly impacting crop yield and quality. Traditional chemical pesticides, due to their advantages of high efficiency, rapid action, broad spectrum, low cost, and ease of use, have long been the primary means of disease control. However, the long-term and excessive use of chemical pesticides easily leads to pesticide resistance in pathogens and causes problems such as pesticide residues and environmental pollution.

[0003] In recent years, the discovery and utilization of plant-derived disease-resistant genes have provided new strategies for crop disease control. Studies have shown that osmotin-like proteins, formed during long-term plant evolution, not only participate in abiotic stress responses, but their conserved chitin-binding sites can also specifically disrupt pathogen cell walls. Currently, antimicrobial osmotin-like proteins have been isolated from model plants such as Arabidopsis thaliana (AtOsm34) and potato (StOsm). These proteins are basic secretory proteins with a molecular weight of approximately 24–26 kDa, containing eight disulfide bonds, an N-terminal signal peptide (mediating endoplasmic reticulum membrane transport), and a C-terminal vacuolar targeting domain, but lacking DNA-binding motifs and glycosylation sites. Plant osmotin-like proteins were initially isolated from tobacco cells under osmotic stress. They belong to the PR-5 family of pathogenesis-related proteins, can be expressed throughout the entire plant, and are one of the important mechanisms for plants to resist microbial invasion. Studies have confirmed that PR-5 proteins can induce programmed cell death in fungi, inhibit their infection process, and activate plant defense signaling pathways, thereby enhancing stress resistance. Permeabilin genes reported in various plants have exhibited broad-spectrum activity in in vitro antibacterial experiments, inhibiting spore germination and hyphal growth of multiple plant pathogens. Their mechanism of action primarily targets the cell membrane of pathogenic microorganisms, exerting antibacterial effects at micromolar concentrations, and is harmless to plant and animal cells, as well as humans and livestock. Given the highly efficient and broad-spectrum antibacterial properties of plant permeabilins, they show great promise for the development of novel biopesticides and antitumor drugs, and are of great significance to sustainable agricultural development and human health.

[0004] Coffee, as the world's leading beverage crop, holds a leading position in terms of yield, value, and consumption. However, its growth is frequently threatened by various pests and diseases, such as coffee rust, berry disease, wilt, anthracnose, as well as stem borers and bark beetles, leading to severe yield reductions and quality declines, causing significant economic losses to the industry. Currently, chemical control remains the primary method of pest and disease control, but its negative impacts on the environment and ecology are becoming increasingly prominent. Therefore, developing green and efficient pest and disease control technologies is crucial for the sustainable development of the coffee industry. As an important tropical economic crop, the coffee genome contains abundant disease-resistant gene resources, which have not yet been systematically explored and utilized. Although existing research has revealed the regulatory function of coffee leaf osmotic proteins in salt stress response, their mechanisms of action in pathogen defense, especially their potential for cross-species application, still require further investigation. Summary of the Invention

[0005] In view of this, the present invention provides a coffee reverse osmosis protein gene and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a coffee reverse osmosis protein gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a coffee reverse osmosis protein encoded by the coffee reverse osmosis protein gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a biomaterial comprising the nucleotide sequence shown in SEQ ID NO.1; the biomaterial is a recombinant vector, cell or microorganism.

[0009] The present invention also provides primer pairs for amplifying the coffee reverse osmosis protein gene, the nucleotide sequences of which are shown in SEQ ID NO.3~4.

[0010] This invention also provides the application of the coffee reverse osmosis protein gene, coffee reverse osmosis protein, or biomaterial in the preparation of antifungal products.

[0011] Preferably, the fungus is one or more of the following: coffee anthracnose fungus, sugarcane red rot fungus, and tea leaf blight fungus.

[0012] It contains at least the following beneficial technical effects: The coffee reverse osmosis protein gene sequence and protein sequence involved in this invention are significantly different from the published coffee reverse osmosis protein OLP gene (NCBI sequence number: XM_027320616.1). The nucleotide sequence length of the coffee reverse osmosis protein gene is 753 bp and the amino acid sequence length is 251, both of which are shorter than OLP. The coffee reverse osmosis protein obtained by this invention has good antibacterial biological activity, can inhibit the growth of coffee anthracnose, and can be used to prepare antibacterial products. It has practical value in the prevention and control of crop diseases such as coffee anthracnose, sugarcane red rot, and tea leaf blight. Attached Figure Description

[0013] Figure 1 The results of agarose gel electrophoresis verification of the coffee reverse osmosis protein prokaryotic expression vector of this invention are shown.

[0014] Figure 2 To illustrate the inhibitory effect of the coffee reverse osmosis protein of this invention on coffee anthrax, the culture medium on the left side of the figure is a blank control without coffee reverse osmosis protein in the equilibration buffer, and the culture medium on the right side shows the inhibitory effect of the coffee reverse osmosis protein of this invention on coffee anthrax.

[0015] Figure 3 To illustrate the inhibitory effect of the coffee reverse osmosis protein of this invention on sugarcane red rot pathogen, the left side of the figure shows a blank control containing a balanced buffer without coffee reverse osmosis protein, while the right side shows the inhibitory effect of the coffee reverse osmosis protein of this invention on sugarcane red rot pathogen.

[0016] Figure 4 To illustrate the inhibitory effect of the coffee reverse osmosis protein of this invention on *Tea leaf blight pathogen*, the left side of the figure shows a blank control containing a balanced buffer without coffee reverse osmosis protein, while the right side shows the inhibitory effect of the coffee reverse osmosis protein of this invention on *Tea leaf blight pathogen*. Detailed Implementation

[0017] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0018] Example 1: Obtaining the Coffee Reverse Osmosis Protein Gene ① Using the published coffee reverse osmosis protein gene OLP (NCBI sequence number: XM_027320616.1) as a reference sequence, a Blast comparison was performed in the coffee transcriptome database (SRA accession: RPJNA847134) to obtain the DNA fragment shown in SEQ ID NO.1.

[0019] Nucleotide sequence information shown in SEQ ID NO.1: .

[0020] ② Using the DNA fragment shown in SEQ ID NO.1 as a template, primers were designed, and its genome sequence was amplified by PCR to verify the accuracy of the transcriptome sequencing results. The primer sequences are as follows: Forward primer (shown in SEQ ID NO.3): 5'-CTGTTTAGCCTGTGTATTCT-3'; Reverse primer (shown in SEQ ID NO.4): 5'-TTTCAGTTCACGCGGGGCAC-3'.

[0021] The 20 μl reaction system includes: 10 μL EasyTaq® PCR SuperMix (TransGold, China), 1 μL each of 10 pmol forward and reverse specific primers, 1 μL coffee DNA, and 7 μL ddH2O.

[0022] The reaction program was as follows: denaturation at 95℃ for 5 min, followed by 35 cycles of amplification at 95℃ for 30 s, 55℃ for 45 s, and 72℃ for 1 min, and then 72℃ for 10 min.

[0023] ③The PCR product was subjected to Sanger sequencing by Guangzhou BGI Biotechnology Co., Ltd., and the sequencing results were consistent with the sequence shown in SEQ ID NO.1.

[0024] ④ The coffee anthracnose pathogen, *Anthracnose*, was inoculated using the puncture inoculation method. A control treatment involved only puncture wounds without pathogen inoculation. Samples were collected 5 days after inoculation. RNA was extracted from the samples using the Tiangen Plant Total RNA Extraction Kit (Tiangen Biotech, Beijing, China) and reverse transcribed into cDNA using the GoScript™ Reverse Transcription System (Promega, USA). The coffee reverse osmosis protein gene was verified using real-time quantitative PCR with the QuantStudio 6 real-time fluorescence quantitative PCR system (Thermo Fisher Scientific, USA), using the same primer sequences as in ②.

[0025] The amplification system consisted of 20 μL, including 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of cDNA template, 10 μL of TransStart Tip Green qPCR Supermix (TransGold, China), 0.4 μL of Passive Reference Dye (TransGold, China), and 7.6 μL of double-distilled water.

[0026] The reaction program was 94℃ for 30 s; 94℃ for 3 min, 94℃ for 10 s, 55℃ for 30 s, for a total of 40 cycles; the dissolution curve was cycled.

[0027] With coffee PP2A The gene (protein phosphatase 2A) was used as an internal control gene. Each sample was replicated three times as a technical replicate. The results were used as 2^30. ΔΔCt Relative quantitative analysis was performed, and the results showed that the coffee reverse osmosis protein gene was significantly upregulated after infection with coffee anthracnose, indicating that it is involved in the coffee disease resistance response.

[0028] Example 2: Cloning and Expression of Coffee Reverse Osmosis Protein Gene ① The entire genome was synthesized by Beijing Qingke Biotechnology Co., Ltd., and then introduced into pEASY. ® -Blunt E1Expression Vector, a prokaryotic expression vector.

[0029] ② The prokaryotic expression vector was introduced into BL21(DE3) Chemically Competent Cells using the heat shock method. After incubation on LB solid medium plates for 12 hours, single clones were picked for PCR detection and LB liquid medium culture.

[0030] ③ Primers were designed using the DNA fragment shown in SEQ ID NO.1 as a template, and PCR was used to detect whether it was inserted into the prokaryotic expression vector. The primer sequences were the same as those in Example 1.

[0031] The 20 μL reaction system includes: 10 μL EasyTaq® PCR SuperMix (Truly Gold, China), 1 μL each of 10 pmol forward and reverse specific primers, 1 μL bacterial culture containing the prokaryotic expression vector, and 7 μL ddH2O.

[0032] The reaction program was as follows: denaturation at 95℃ for 5 min, followed by 35 cycles of amplification at 95℃ for 30 s, 55℃ for 45 s, and 72℃ for 1 min, and then 72℃ for 10 min.

[0033] ④ Agarose gel electrophoresis showed that the PCR product was approximately 750 bp in length (see...). Figure 1 The results were as expected. The PCR product was sequenced by Sanger sequencing at Guangzhou BGI Biotechnology Co., Ltd., and the sequencing results were consistent with the sequence shown in SEQ ID NO.1.

[0034] ⑤ BL21 competent cells containing the prokaryotic expression vector were cultured in LB liquid medium for 24 hours, and after being sonicated, coffee reverse osmosis protein was extracted using the GST NUPharose FF pre-packed column protein purification kit (Sangon Biotech, China).

[0035] Amino acid sequence information of coffee reverse osmosis protein (SEQ ID NO.2): MATLPFPAMLSLSLLFSLCILSTATQPGMILTLVNNCPYTVWPAIQPNAGHPVLERGGFALHTLTHRSFAAPTTHWSGRIWARTGCTYSNNHFSCATGDCGGRIECDGRGGATPATLVQLVLHHG PADFSSYGVSLVDGFNIPMTVTPHEGKGTCPVVGCRVDLVPTCPASLRFHGPGGHVVGCKSGCEAFGTDELCCRNHYNSKETCKPSSYSDYFKHACPATFTYAHDSPSLMHECSAPRELKVIFCH.

[0036] Example 3 Antibacterial Activity of Coffee Reverse Osmosis Protein Coffee reverse osmosis protein was prepared into a 100 μM test solution using equilibration buffer PBS (300 mM NaCl, 50 mM NaH2PO4, 10 mM Mimidazole, 10 mM Tris base, pH 8.0) as the solvent. Antibacterial activity was tested using the Oxford cup method. *Anthracis aureus*, *Coffea spp.*, and *Tea spp.* were used as indicator bacteria. Their bacterial blocks were inoculated in the center of a PDA plate, and Oxford cups were placed around the blocks. 100 µL of the protein test solution or an equal volume of equilibration buffer PBS was added as a blank control. Colony growth was observed after incubation at 28°C for 5-7 days.

[0037] The results are as follows Figures 2-4 As shown, the equilibration buffer is effective against coffee anthrax ( Figure 2 Left), sugarcane red rot fungus ( Figure 3 Left), tea leaf blight pathogen ( Figure 4 Left) has no inhibitory effect; the balanced buffer containing caffeine reverse osmosis protein inhibits the growth of coffee anthrax bacteria ( Figure 2 (Right), sugarcane red rot fungus ( Figure 3 Right), tea leaf blight colony ( Figure 4 (Right) An arc-shaped inhibition zone is generated at the edge of the Oxford cup, indicating that the coffee reverse osmosis protein has an inhibitory effect on coffee anthracnose, sugarcane red rot, and tea leaf blight. This indicates that the coffee reverse osmosis protein of the present invention can be used to prepare antibacterial products and applied to the prevention and control of coffee anthracnose, sugarcane red rot, and tea leaf blight.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A coffee reverse osmosis protein gene, characterized in that, The nucleotide sequence of the coffee reverse osmosis protein gene is shown in SEQ ID NO.

1.

2. A coffee reverse osmosis protein encoded by the coffee reverse osmosis protein gene according to claim 1, characterized in that, The amino acid sequence of the coffee reverse osmosis protein is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The nucleotide sequence shown in SEQ ID NO.1 is introduced into the biological material; the biological material is a recombinant vector, cell, or microorganism.

4. The primer pair for amplifying the coffee reverse osmosis protein gene according to claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.3~4.

5. The use of the coffee reverse osmosis protein gene of claim 1, the coffee reverse osmosis protein of claim 2, or the biomaterial of claim 3 in the preparation of antifungal products.

6. The application according to claim 5, characterized in that, The fungus is one or more of the following: coffee anthracnose fungus, sugarcane red rot fungus, and tea leaf blight fungus.

Images