Glomus huanghuai GIP12 effect protein as well as coding gene and application of glomus huanghuai GIP12 effect protein

Abstract

The invention belongs to the technical field of gene engineering, and discloses a glomus huanghuai GIP12 protein, a coding gene and application thereof, the amino acid sequence of the glomus huanghuai GIP12 effect protein is shown as SEQ ID NO.2, and the nucleotide sequence of nucleic acid or the gene for coding the effect protein is shown as SEQ ID NO.1. The invention further discloses a preparation method of the glomus huanghuai GIP12 protein. The invention proves that the glomus huanghuai GIP12 protein plays an important role in the process of improving the disease resistance of plants through cloning the glomus huanghuai GIP12 protein gene and transient expression of tobacco.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a GIP12 effector protein of Hymenococcus huaiense, its encoding gene and its application. Background Technology

[0002] Throughout their life cycle, plants are attacked by various microbial pathogens. In the process of co-evolution between plants and pathogens, adaptive pathogens have evolved various effector proteins to attack plants. Avr effectors are a class of proteins that can be directly or indirectly recognized by plant R proteins, triggering a hypersensitive response (HR). The purpose of the HR response is to limit the further spread and infection of pathogens. Plants form physical barriers through cell death to prevent the spread of pathogens and secrete antimicrobial substances and reactive oxygen species to inhibit pathogen growth. This also serves as an important signal for stimulating plant immunity.

[0003] Glucose-dependent insulinotropic polypeptide effector proteins (GIPs) are a family of secreted proteins unique to oomycetes (such as Phytophthora and Downy mildew), encoding bifunctional proteins containing specific functional domains and signal regulation modules. During infection, GIPs regulate host glucose metabolism and immune homeostasis by targeting key signaling pathways within host cells, creating a suitable infection microenvironment for pathogens. Simultaneously, as effectors, they are recognized by the plant immune system, triggering complex immune responses (hormone signal reprogramming, regulation of defense gene expression, and cellular metabolic remodeling). Due to their core regulatory role in oomycete pathogenicity strategies and host immune escape, GIPs have become a key target for elucidating the molecular mechanisms of oomycete-plant interactions and are being explored as potential target proteins for novel crop disease control strategies. Currently, no reports have been found in *Cyclocarya paliurus*. *Cyclocarya paliurus* (Huanghuai) Globisporangium huanghuaiense (This is a pathogenic oomycete that can cause root rot in plants such as wheat, rice, and soybeans, resulting in serious economic losses.) Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide a GIP effector protein from *Hypericum huanghuaiense* that can induce a hypersensitive response (HR) in plants. The second objective is to provide the nucleic acid or gene encoding the aforementioned *Hypericum huanghuaiense* GIP 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 *Hypericum huanghuaiense* GIP effector protein, nucleic acid or gene, expression cassette, recombinant expression vector, transgenic cell line, or transgenic recombinant bacteria.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The present invention relates to the GIP12 effector protein of *Cyclocarya paliurus*, wherein the amino acid sequence of the GIP12 effector protein is shown in SEQ ID NO.2.

[0006] The present invention relates to a nucleic acid or gene encoding the above-mentioned GIP12 effector protein of *Cyclocarya paliurus*, wherein the nucleotide sequence of the nucleic acid or gene is shown in SEQ ID NO.1.

[0007] The present invention relates to expression cassettes, recombinant expression vectors, transgenic cell lines, or transgenic recombinant bacteria containing the above-mentioned *Cyclocarya paliurus* GIP12 effector protein.

[0008] The present invention relates to a method for constructing a recombinant expression vector, wherein the expression vector is obtained by introducing the gene encoding the GIP12 effector protein of *Hylocereus huanghuaiense* into the plant expression vector pCAMBIA1300-FLAG.

[0009] The nucleotide sequences of the primers used in the construction of the expression vector are shown in SEQ ID NO.3~4.

[0010] 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.

[0011] The *E. coli* strain is a DH5α competent cell.

[0012] The present invention relates to the application of the above-mentioned *Hylocereus huanghuaiense* GIP12 effector protein, 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.

[0013] The present invention relates to the application of the above-mentioned *Hylocereus huanghuaiense* GIP12 effector protein, nucleic acid or gene, expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria in the preparation of plant resistance reagents.

[0014] The improvement of plant resistance refers to improving the plant's resistance to infection by Phytophthora capsici.

[0015] Beneficial effects: Compared with existing technologies, this invention has the following significant advantages: Through extensive screening, this invention obtained a gene GIP12 derived from *Hylocereus huanghuaiense* that can trigger a plant hypersensitive response (HR). This gene triggers plant immunity and induces disease resistance, which is of great significance for developing environmentally friendly disease resistance strategies. Attached Figure Description

[0016] Figure 1 : GIP12 Phenotypic diagram of the HR effect in gene-induced plants.

[0017] Figure 2 GIP12 triggers HR to induce plant resistance. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0019] Example 1: GIP effector protein gene GIP12 Amplification and sequencing 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℃.

[0020] 2. Preparation of the tested tobacco seedlings 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.

[0021] 3. Extraction of *Cyclocarya paliurus* from the Huanghuai region ( 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.

[0022] Table 1 Primer Sequences Name Sequence 5’ - 3’ PYHUGIP12-F GACGAGCTGTACAAGGGTACCATGAAGGTCCTCCAAGCGTTC PYHUGIP12-R GCGGACTCTAGTTCATCTAGAACGCCAGGTGGCGGCAGG 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.

[0023] The PCR reaction procedure was as follows: pre-denaturation at 95℃ for 5 min; 95℃ pre-denaturation ... Denaturation at 55℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, 30 cycles; extension at 72℃ for 4 min; store at 12℃.

[0024] 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 GIP protein gene. This GIP protein gene is derived from *Cyclocarya paliurus* (Huanghuai). Globisporangium huanghuaiense GIP protein gene GIP12 The GIP12 The nucleotide sequence of the gene is shown in SEQ ID NO.1. GIP12 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0025] SEQ ID NO.1 ATGAAGGTCCTCCAAGCGTTCGCTGCCTCGGCCGTCCTCGCCACCGCCTCCACCATCGCCGCCGTCGACGCGCGCGCCGGACCGATCACCTTCGAGGAGTACATGACCGACATCACCCTGGTCAACTGGGAGGCTGAGAACGCCATCACGCCGCTAATCCTCGGTGGCACCGAGACCCCGGTCGGCACCTCCACGTACGTGGCCGGCATGCGCAGCACCGCCGCCGGCTCCAGCTTCTGCGGTGGCTCGCTCATCGCGCCGACGTGGATCCTCACGGCCGCGCACTGCGCCAGCAGCATCCAGTACGTCAACGTCGGCACGCACTACCTGTCGGGGACCACGGACGGCACGGCCGTCAAGGTGCTGCGCAAGATCATCCACCCGCTCTACAAGAGCGCCAGCACGGGCAACGACTTCCTCCTGCTCGAGCTCGCCTCGCCCGTGTCCTACCCACCAGTGGTGCTCGCGGCCGCCAACGGCACGGACGAGACCGTGGGGAGCGTCGCCACCACACTCGGATGGGGGACTACCACCTCAGGCGGCTCCCAGTCGCGCGTGCTGCTCCAGGTCGACGTCGCCATCGTGACCAACGCGGAGTGCAAGGCCAAGCTCAGTAGCGTCACGGACACCATGATCTGCGCCGGTGGACTCTCGAACAAGGACTCGTGCCAGGGCGACTCGGGCGGTCCACTCGTCACCAAGCAGAACGCCACGGACGTGCTCGTGGGCGTCGTCAGCTGGGGCCGCGGCTGCGGCCAGCTCGGCTACCCAGGCGTCTACTCGCGCGTCTCGGCCGGCCGCGCCTTCATCGACCAGTACGTGCCTGCCGCCACCTGGCGTTAG SEQ ID NO.2 MKVLQAFAASAVLATASTIAAVDARAGPITFEEYMTDITLVNWEAENAITPLILGGTETPVGTSTYVAGMRSTAAGSSFCGGSLIAPTWILTAAHCASSIQYVNVGTHYLSGTTDGTAVKVLRKIIHPLYKSASTGNDFL LLELASPVSYPPVVLAAANGTDETVGSVATTLGWGTTTSGGSQSRVLLQVDVAIVTNAECKAKLSSVTDTMICAGGLSNKDSCQGDSGGPLVTKQNATDVLVGVVSWGRGCGQLGYPGVYSRVSAGRAFIDQYVPAATWR* Example 2: Functional Verification of GIP12 1. Construct the pCAMBIA1300-FLAG:GIP12 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 GIP12 (SEQ ID NO.1) was ligated to the vector using the homologous recombinase from Novizan to obtain the pCAMBIA1300-FLAG:GIP12 recombinant vector. The pCAMBIA1300-FLAG:GFP and pCAMBIA1300-FLAG:INF1 vectors were pre-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.).

[0026] The reaction system is as follows: 1 μL 10×CE II reaction buffer; 2 μL PCR purified product of GIP12; 1 μL of the above-digested pCAMBIA1300-FLAG empty vector; 5 μL homologous recombinase; and sterile water to a final volume of 10 μL.

[0027] 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. After completion, 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:GIP12 expression vector was obtained.

[0028] 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:GIP12. Add 30 ng of pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:GIP12 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℃ for 5 min, and place on ice for 5 min. After incubation, add 700 μL of fresh LB broth and incubate at 28℃ 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℃ for 48 h. Pick single colonies for colony PCR to obtain positive transformants.

[0029] 2.2 Transient expression of pCAMBIA1300-FLAG:GFP, pCAMBIA1300-FLAG:INF1, and pCAMBIA1300-FLAG:GIP12 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.

[0030] 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:GIP12 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 100 μL of each combination was injected. Leaf phenotypes were observed for 7 days for any significant hazard ratio (HR). Each combination was repeated in triplicate. 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... GIP12 Genes can trigger HR responses.

[0031] 2.3 Detection of resistance to Nicotiana benthamiana with transient expression of pCAMBIA1300-FLAG:GIP12 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 GIP12 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 of ​​the Phytophthora capsici infection zone, where pCAMBIA1300-FLAG:GIP12 protein was injected, is 138 mm². 2 In the control group, the infection area of ​​Phytophthora capsici was 213 mm². 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 GIP12 protein from *Hylocereus huanghuaiensis* significantly improved the resistance of *Nicotiana benthamiana* to *Phytophthora capsici*.

Claims

1. A GIP12 effector protein from *Cyclocarya paliurus*, characterized in that, The amino acid sequence of the GIP12 effector protein of *Cyclocystis huanghuaiensis* is shown in SEQ ID NO.

2.

2. The nucleic acid or gene encoding the GIP12 effector protein 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 bacterium containing the nucleic acid or gene of the *Cyclocarya paliurus* GIP12 effector protein 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 GIP12 effector protein 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 use of the *Hylocereus huanghuaiense* GIP12 effector protein of claim 1, the nucleic acid or gene of claim 2, the expression cassette of claim 3, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacteria in the preparation of reagents for inducing plant allergic reactions.

9. The application of the *Hylocereus huanghuaiense* GIP12 effector protein of claim 1, the nucleic acid or gene of claim 2, the expression cassette of claim 3, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacteria 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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