Endosporium huanghuai effect protein EPI3 as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a glomus huanghuai effect protein EPI3 protein, and coding and application thereof, the amino acid sequence of the glomus huanghuai effect protein EPI3 protein is shown as SEQ ID NO.2, and the nucleotide sequence for coding the gene is shown as SEQ ID NO.1. The invention further discloses a preparation method of the glomus huanghuai effect protein EPI3 protein. The invention proves that the glomus huanghuai EPI3 protein plays an important role in the process of improving the disease resistance of plants through cloning of the glomus huanghuai EPI3 protein gene and transient expression of tobacco.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an effector protein EPI3 of *Cyclocarya paliurus*, its encoding gene, and its applications. Background Technology

[0002] Huanghuai Coccidioidomyces ( Globisporangium huanghuaiense The same thing is called Huanghuai Pythium ( Pythium huanghuaiense (This is a soil-borne pathogen that can cause root rot and damping-off in various crops such as wheat, rice, and soybeans, leading to reduced seedling emergence, stunted plant growth, and even death, causing serious economic losses to agricultural production.)

[0003] During infection, some plant pathogens can secrete various effector proteins into the host tissue. These proteins are key factors in regulating plant immune responses and can be mainly divided into two categories based on their sites of action: apoplast effector proteins (acting in the extracellular space of plant cells) and cytoplasmic effector proteins (entering plant cells to exert their effects). Based on their functions, effector proteins can be divided into elicitors (which can induce plant immune responses) and repressors (which suppress plant immunity), playing opposite roles in pathogen-host interactions. Elicitors can be recognized by pattern recognition receptors on the surface of plant cells, thereby activating "pattern-triggered immunity" (PTI). Due to their ability to induce plant resistance and their environmental friendliness, elicitors have become an important direction in the research and development of alternatives to traditional chemical pesticides.

[0004] Extracellular Protease Inhibitors (EPIs) are a class of pathogenic effector molecules identified from the secretome of oomycetes such as *Phytophthora*. Their protein sequences encode a signal peptide at the N-terminus, followed by a tandem sequence of Kazal-type domains with protease inhibitory activity. EPIs are widely present and functionally conserved in *Phytophthora*, but no related functions have been reported in *Cyclocarya*. Elucidating the immune activation mechanism of EPIs and developing recombinant EPI proteins capable of efficiently triggering plant hypersensitive responses (HRs) will enable the rapid initiation of plant immune responses through HR stimulation, inducing broad-spectrum disease resistance. This has significant theoretical value and application prospects for developing environmentally friendly oomycete disease control strategies. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide an EPI effector protein from *Hypersensitive phytoreceptor* that can induce a hypersensitive response (HR) in plants. The second objective is to provide the nucleic acid or gene encoding the aforementioned *Hypersensitive phytoreceptor* EPI effector protein. The third objective is to provide an expression cassette, recombinant expression vector, transgenic cell line, or transgenic recombinant bacteria containing the aforementioned nucleic acid or gene, as well as methods for constructing the recombinant expression vector and transgenic recombinant bacteria. The fourth objective is to provide applications of the aforementioned *Hypersensitive phytoreceptor* EPI effector protein, nucleic acid or gene, expression cassette, recombinant expression vector, transgenic cell line, or transgenic recombinant bacteria.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention relates to the Huanghuai coccidioidomyces effector protein EPI3, wherein the amino acid sequence of the Huanghuai coccidioidomyces effector protein EPI3 is shown in SEQ ID NO.2.

[0007] The present invention relates to the nucleic acid or gene encoding the above-mentioned Huanghuai cysticercosis effector protein EPI3, wherein the nucleotide sequence of the nucleic acid or gene is shown in SEQ ID NO.1.

[0008] The present invention relates to expression cassettes, recombinant expression vectors, transgenic cell lines, or transgenic recombinant bacteria containing the above-mentioned Huanghuai cysticercosis effector protein EPI3.

[0009] The present invention discloses a method for constructing a recombinant expression vector, wherein the expression vector is obtained by introducing the gene encoding the EPI effector protein of *Hylocereus huanghuaiense* into the plant expression vector pCAMBIA1300-FLAG.

[0010] The nucleotide sequences of the primers used in the construction of the expression vector are shown in SEQ ID NO.3~4.

[0011] The transgenic recombinant bacteria of the present invention are obtained by inserting the above-mentioned recombinant expression vector into Escherichia coli and screening for transgenic recombinant bacteria.

[0012] The *E. coli* strain is a DH5α competent cell.

[0013] The present invention relates to the application of the above-mentioned Huanghuai cysticercosis effector protein EPI3, nucleic acid or gene, expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria in the preparation of reagents for inducing plant allergic reactions.

[0014] The present invention relates to the application of the above-mentioned Huanghuai cysticercosis effector protein EPI3, nucleic acid or gene, expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria in the preparation of a reagent to improve plant resistance.

[0015] The improvement of plant resistance refers to improving the plant's resistance to infection by Phytophthora capsici.

[0016] Beneficial effects: Compared with existing technologies, this invention has the following significant advantages: Through extensive screening, this invention obtained a gene EPI3 derived from *Hylocereus huanghuaiense* that can trigger a plant hypersensitive response (HR). This gene triggers plant immunity and induces disease resistance in plants, which is of great significance for developing environmentally friendly disease resistance strategies. Attached Figure Description

[0017] Figure 1 : EPI3 Phenotypic diagram of the HR effect in gene-induced plants.

[0018] Figure 2 EPI3 triggers HR to induce plant resistance. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0020] Example 1: Amplification and sequencing of the EPI effector protein gene EPI3 1. Experimental strains The tested strain was *Cyclocarya huanghuai* ( Globisporangium huanghuaiense The strain was preserved by the Green Control Laboratory of Jiangsu Vocational College of Agriculture and Forestry. The strain was preserved on 10% V8 solid slant at a temperature of 10℃.

[0021] 2. Preparation of seedlings for testing *Nicotiana benthamiana* Ben's tobacco ( Nicotiana benthamiana It is placed in a plant growth chamber with 16 hours of light and 8 hours of darkness and 70% relative humidity.

[0022] 3. Extraction of *Cyclocarya paliurus* (Huanghuai) Globisporangium huanghuaiense RNA was extracted, and cDNA of the target gene was obtained. Hyphae of *Cyclocarya paliurus* were collected, flash-frozen in liquid nitrogen, and then cooled and ground in a mortar. Total RNA was extracted from *Cyclocarya paliurus* according to the recommended method of the Novizan Total RNA Extraction Kit (catalog number: R711-01). cDNA was synthesized according to the method provided by the Novizan RNA Reverse Transcription Kit (catalog number: R212-01). Using the above cDNA as a template, PCR amplification was performed with the corresponding forward and reverse primers (SEQ ID NO. 3~4). The primer sequences are shown in Table 1.

[0023] Table 1 Primer Sequences Name Sequence PyhuEPI3-F GACGAGCTGTACAAGGGTACCATGGCAAAGATCTGCAAGCC PyhuEPI3-R GCGGACTCTAGTTCATCTAGACTCAGTCAACGCGATCGCC The PCR reaction system consisted of: 12.5 μL high-fidelity enzyme; 0.5 μL primer; 0.5 μL template; and sterile water to a final volume of 25 μL.

[0024] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 4 min; and storage at 12℃.

[0025] The amplified PCR products were subjected to 1% agarose gel electrophoresis and observed under UV light. PCR products showing the target band were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. After confirmation by sequencing, the obtained sequence was found to be the nucleotide sequence of the EPI protein gene. This EPI protein gene is derived from *Cyclocarya paliurus* (Huanghuai). Globisporangium huanghuaiense EPI protein gene EPI3 The EPI3 The nucleotide sequence of the gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the EPI3 gene is shown in SEQ ID NO.2.

[0026] SEQ ID NO.1 ATGGCAAAGATCTGCAAGCCGTACTCGCTATTCATGGCTGCTGCCGCGCTCGCACTCTTCGCATCGTCCTCGACAATGAACTCCGTCGCTGCGCAAGAAGACTCCGGCATTGAGCTCGTGAACCTGGATGCTGACATTGTCGGCGCTGTCGCTGCCCCCAGTGACGATTTGGTCGGAAACGAAGCCCCTGCGAGCAGCTACACTGGTGACGAGACTGCGGCGCCAACTGAAGAGGTCGCAGGAGAAGCTGGTACGCCAGTGGATACTACCGATGCTGCGCCAAGCTCGGATGTCACAGAAGAAGCCCGATCTGCTGACGAATTGGCTCAGAGCGCTAACGAGGCTGCTGTCCCAGAAGAAGTCCGCTCCGACGATGCTGCGGCTGCAGCTGCCGATGAATCTGTCACAGAGAGCGGCGATGCTGTGCCAACGGAGGACGTTGCCAGCGACGCGGACGCAACCACTGTCGGTGCTGCTGACGATACGCCTGGAAGTGAAGCCACTGCTGTTACTGATGCCACAGCCAACGACATTGTCGGAAGTGATGCCACGATTCTGGGTGGCGCGACGATCCTCGGCAAGTCGCAAGAGCTGGAACTCAATGACGCCGCCGTGTCAGTGCTTGTCGACGCTCTGAGCACGCCAACCAACTACGACCCGACCATCACAGCGCCTATCTGCGTGCTGCAGATCAACAGCGCACAGTTCCAGACCGTATCCGGCACGAACTACCGCTACCAGGTGCTGGGGTGCGCGATCAACTTCGCCGACGAGCTTGGCGCGTGCCGCAACCGCCAGTGCACGCAGGCGCCGTACGAAGTCACGATCTACCAGCAGACGTGGACCAACACGCTGCAGGTTTCGGCGATCGCGTTGACTGAGTAG SEQ ID NO.2 MAKICKPYSLFMAAAALALFASSSTMNSVAAQEDSGIELVNLDADIVGAVAAPSDDLVGNEAPASSYTGDETAAPTEEVAGEAGTPVDTTDAAPSSDVTEEARSADELAQSANEAAVPEEVRSDDAAAAAADESVTESGDAVPTEDV ASDADATTVGAADDTPGSEATAVTDATANDIVGSDATILGGATILGKSQELELNDAAVSVLVDALSTPTNYDPTITAPICVLQINSAQFQTVSGTNYRYQVLGCAINFADELGACRNRQCTQAPYEVTIYQQTWTNTLQVSAIALTE* Example 2: Functional Verification of EPI3 1. Construct the pCAMBIA1300-FLAG:EPI3 expression vector The empty vector plasmid pCAMBIA1300-FLAG (BIOVECTOR China Plasmid Vector Strain Cell Line Gene Preservation Center) was digested with KpnⅠ and SalⅠ enzymes. The target fragment EPI3 (SEQ ID NO.1) was ligated to the vector using the homologous recombinase from Novizan to obtain the pCAMBIA1300-FLAG:EPI3 recombinant vector. The pCAMBIA1300-FLAG:GFP and pCAMBIA1300-FLAG:INF1 vectors were already constructed in the laboratory using conventional methods disclosed in existing technologies (refer to Chen R, Ma D, Bao Y, et al. Joint application of plant immunity-inducing elicitors and fungicides to control Phytophthora diseases[J]. Phytopathology Research, 2024, 6(1):15.DOI:10.1186 / s42483-024-00233-0.).

[0027] The reaction system is as follows: 1 μL 10×CE II reaction buffer; 2 μL PCR purified product of EPI3; 1 μL of the above-digested pCAMBIA1300-FLAG empty vector; 5 μL homologous recombinase; and sterile water to a final volume of 10 μL.

[0028] The above reaction system was reacted at 37 ℃ for 30 min. The ligation product was then transformed into 100 μL of *E. coli* DH5α competent cells, incubated at 42 ℃ for 1 min, and placed on ice for 2 min. Afterward, 700 μL of fresh LB broth was added, and the cells were incubated at 37 ℃ with shaking for 1 h. After incubation, the cells were centrifuged at 5000 rpm for 3 min. 100 μL of the bacterial culture was plated onto a solid LB agar plate containing 50 mg / mL kanamycin, and incubated upside down at 37 ℃ for 16 h. Single colonies were then picked for colony PCR identification. The identified positive transformants were sequenced; if correct, the pCAMBIA1300-FLAG:EPI3 expression vector was obtained.

[0029] 2. Transformation of expression vector into Agrobacterium and functional verification 2.1. Agrobacterium was transformed with expression vectors pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:EPI3. Add 30 ng of pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:EPI3 expression vectors to 100 μL of Agrobacterium GV3101 competent cells, respectively. Incubate on ice for 5 min, freeze in liquid nitrogen for 5 min, incubate at 37°C for 5 min, and place on ice for 5 min. After incubation, add 700 μL of fresh LB broth and incubate at 28°C with shaking for 2 h. After incubation, centrifuge at 5000 rpm for 3 min. Retain 100 μL of bacterial culture and plate it onto a solid LB agar plate containing 50 mg / mL kanamycin and 25 mg / mL rifampicin. Incubate upside down at 28°C for 48 h. Pick single colonies for colony PCR to obtain positive transformants.

[0030] 2.2 Transient expression of pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:EPI3 proteins in Nicotiana benthamiana. Tobacco Benedict ( Nicotiana benthamiana The plants were placed in a plant growth chamber with 16 hours of light and 8 hours of darkness and 70% relative humidity. After 30 days, leaves from the same part were used for gene expression experiments.

[0031] The positive transformants were placed in LB liquid medium and cultured at 28°C with shaking for 30 h. The bacterial culture was collected, centrifuged at 8000 rpm for 2 min, and washed three times with 10 mM MgCl2. Finally, the bacterial culture was diluted with 10 mM MgCl2 to an OD 600 value of 0.3. Bacterial cultures containing pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:EPI3 were mixed 1:1 with Agrobacterium GV3101 culture (OD 600 = 0.3) containing the plant gene silencing repressor p19 protein of tomato dwarf virus (which can significantly enhance the transient expression efficiency of the target gene). All gene combinations were injected into leaves of Tobacco Benedict using a 1 mL syringe, ensuring that each combination was injected at 100 μL. The leaf phenotype was observed for 7 days to see if there was a significant HR. Each combination was repeated in 5 replicates, and the experiment was repeated three times. The results are shown below. Figure 1 As shown in the figure. The pCAMBIA1300-FLAG:GFP group served as the negative control, and the pCAMBIA1300-FLAG:INF1 group served as the positive control. It is clear that... EPI3 Genes can trigger HR responses.

[0032] 2.3 Detection of disease resistance in *Nicotiana benthamiana* with transient expression of pCAMBIA1300-FLAG:EPI3 protein Leaves expressing the confirmed protein were cut and placed in a filter paper-insulated tray. Holes were punched along the edge of the *Phytophthora capsici* LT263 agar plate using a 7 mm punch, and the fungal discs were placed symmetrically on either side of the *N. benthamiana* leaves, mycelial side down. The trays were placed in a 25°C incubator in the dark for 36 h. The leaves were then removed and irradiated with UV light. The areas that darkened in color were the *Phytophthora capsici* infection areas. The area of ​​the infection area was measured with a ruler. Leaves expressing pCAMBIA1300-FLAG:GFP protein served as controls. The effect of EPI3 protein on resistance in *N. benthamiana* was compared. Results are as follows: Figure 2 As shown, Figure 2 The left image shows a Tobacco Benedict leaf infected with Phytophthora capsici. The area infected with pCAMBIA1300-FLAG:EPI3 protein was 95 mm². 2 In the control group, the infected area of ​​Phytophthora capsici was 198 mm. 2 ; Figure 2 The right side shows the statistical results of lesion area for 15 replicates of Agrobacterium-induced disease resistance phenotypes, which ultimately verified that the EPI3 protein from *Hylocereus huanghuaiensis* significantly improved the resistance of *Nicotiana benthamiana* to *Phytophthora capsici*.

Claims

1. An effector protein EPI3 from *Cyclocarya paliurus*, characterized in that, The amino acid sequence of the effector protein EPI3 from *Cyclocystis jirovecii* is shown in SEQ ID NO.

2.

2. The nucleic acid or gene encoding the effector protein EPI3 of *Cyclocarya paliurus* as described in claim 1, characterized in that, The nucleotide sequence of the nucleic acid or gene is shown in SEQ ID NO.

1.

3. An expression cassette, recombinant expression vector, transgenic cell line, or transgenic recombinant bacteria containing the nucleic acid or gene of the Huanghuai Coccidioidomyces effector protein EPI3 as described in claim 2.

4. The method for constructing the recombinant expression vector according to claim 3, characterized in that, The expression vector was obtained by introducing the gene encoding the effector protein EPI3 of *Hylocereus huanghuaiense* into the plant expression vector pCAMBIA1300-FLAG.

5. The construction method according to claim 4, characterized in that, The nucleotide sequences of the primers used in the construction of the expression vector are shown in SEQ ID NO.3~4.

6. The transgenic recombinant bacteria according to claim 3, characterized in that, The recombinant expression vector described in claim 3 was inserted into Escherichia coli, and transgenic recombinant bacteria were obtained by screening.

7. The transgenic recombinant bacteria according to claim 6, characterized in that, The Escherichia coli were DH5α competent cells.

8. The application of the Huanghuai Coccidioidomyces effector protein EPI3 as described in claim 1, the nucleic acid or gene as described in claim 2, the expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria as described in claim 3 in the preparation of reagents for inducing plant allergic reactions.

9. The application of the Huanghuai Coccidioidomyces effector protein EPI3 as described in claim 1, the nucleic acid or gene as described in claim 2, the expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria as described in claim 3 in the preparation of reagents to enhance plant resistance.

10. The application according to claim 9, characterized in that, The improvement of plant resistance refers to enhancing the plant's resistance to infection by Phytophthora capsici.

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