A polynucleotide, a method for controlling root-knot nematodes of the family olygariae and use thereof

Abstract

This invention discloses a polynucleotide, a method for controlling the root-knot nematode of the Poaceae family, and its application. The polynucleotide contains the nucleotide sequence shown in SEQ ID NO:1, and its encoded effector protein contains the amino acid sequence shown in SEQ ID NO:2. Double-stranded RNA targeting the polynucleotide can control the root-knot nematode of the Poaceae family. This invention utilizes Mg-grn-1 dsRNA for in vitro RNAi against the root-knot nematode of the Poaceae family, effectively reducing the nematode's infectivity. RNAi transgenic rice experiments show that silencing this gene significantly enhances plant resistance, while overexpression increases sensitivity. This invention provides key targets and technical basis for developing novel RNAi biopesticides and breeding nematode-resistant crop varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a polynucleotide, a method for controlling root-knot nematodes of the Poaceae family, and its application. Background Technology

[0002] Rice is the staple food for half the world's population and is crucial for food security. Root-knot nematodes (of the Poaceae family) Meloidogyne graminicola ) is one of the important pathogenic nematodes of rice, which occurs widely in rice-producing areas of Asia and Africa. It causes the formation of hook-shaped root knots in the root tips of rice plants, damages the vascular tissue of the roots, and hinders the transport of water and nutrients, resulting in symptoms such as yellowing, dwarfing, and delayed maturity of the plants, ultimately leading to a yield loss of 16%-97% (Feng Hui et al., 2017).

[0003] Root-knot nematodes of the Poaceae family infect the root system as second-instar larvae, inducing the formation of giant cells, which serve as a dedicated nutrient reservoir to sustain further infection, development, and reproduction. During early interactions with the host plant, second-instar nematodes produce secretory proteins from their esophageal gland cells, which are then secreted into the host plant via their stylets. These proteins generally contain signal peptides but lack transmembrane domains, possessing effector functions. They can inhibit plant immune responses and induce the maintenance of feeding cells by disrupting cell wall barriers and regulating reactive oxygen species bursts, gene transcription, and histone modifications, thereby enhancing the pathogenicity of root-knot nematodes.

[0004] Granulin (GRN) belongs to a conserved family of secretory glycosylation proteins and is widely involved in various pathophysiological activities, including inflammatory responses, damage repair, growth and development, and metabolic regulation [Bowhay CR and Hanington PC. Animal granulins: In the GRN scheme of things. Developmental and Comparative Immunology. 2024, 152: 105115]. Human liver flukes can secrete granulin-like growth hormones that can lead to the development of cholangiocarcinoma [Wang et al. Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma. Parasites & Vectors. 2017, 10:262]. No reports have yet been found regarding granulin from plant parasitic nematodes.

[0005] In recent years, with the release of the genome of the root-knot nematode (Poaceae), research on the functions of its effectors has deepened. Some effectsor functions have been reported; for example, the effector gene MgCL11340 can inhibit reactive oxygen species (ROS) bursts in plants and is localized in the cell margins and nucleus (Chinese Invention Patent CN118652896A); the transcription factor MgBTF3, although lacking a typical secretion signal peptide, can be secreted through non-classical pathways and can enhance nematode infectivity (Chinese Invention Patent CN113801213B); the effector MgMO603 can also promote nematode parasitism, and its corresponding RNAi transgenic rice exhibits resistance (Chinese Invention Patent CN118652318A). However, the current technologies for identifying more key effectors in the infection process of the root-knot nematode and their novel mechanisms in regulating host immunity still face challenges such as limited target genes and unclear mechanisms of action, and no effective solutions have been proposed. A comprehensive search of relevant domestic and international literature and patents has revealed no reports on the effector gene Mg-grn-1 and its function in the root-knot nematode. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a polynucleotide-based method for controlling root-knot nematodes (Poaceae) and its application. This addresses the deficiencies in existing root-knot nematode control techniques and the lack of research on the nematode effector gene Mg-grn-1, and explores its correlation with nematode infection and parasitism. This invention provides a new target for the development of drugs against root-knot nematodes, offers a new approach to their control, and discovers that the effector protein Mg-GRN-1 is a specific particulate protein effector protein of root-knot nematodes, which enhances the pathogenicity of root-knot nematodes by inhibiting plant basal immunity.

[0007] Based on transcriptome sequencing, our laboratory identified a large number of potential effector genes in the early-stage second-instar root-knot nematode larvae of the Poaceae family, including several highly expressed proteins with unknown functions. Analyzing the biological functions of these effectors can provide valuable insights for the prevention and control of rice root-knot nematode disease.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention is to provide a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:1, or a variant having at least 95% sequence identity with SEQ ID NO:1 and encoding a protein having inhibitory plant immune activity.

[0009] Furthermore, the polynucleotide is the Mg-grn-1 effector gene of the root-knot nematode of the Poaceae family.

[0010] A second aspect of the present invention is to provide an effector protein comprising the amino acid sequence shown in SEQ ID NO:2, or a variant having at least 95% sequence identity with SEQ ID NO:2 and having inhibitory plant immune activity.

[0011] Furthermore, the first to 24th amino acids at the N-terminus of the effector protein are a signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3.

[0012] A third aspect of the present invention is to provide a method for controlling root-knot nematodes of the Poaceae family, said method being implemented in any of the following ways: (1) In vitro RNA interference was performed on the root-knot nematode of the Poaceae family using double-stranded RNA targeting the polynucleotides described in the first aspect; (2) Constructing a transgenic plant resistant to root-knot nematodes of the Poaceae family using double-stranded RNA targeting the polynucleotides described in the first aspect, wherein the plant is tobacco or rice, a Poaceae plant.

[0013] Furthermore, the double-stranded RNA is dsRNA, which contains a sequence complementary to at least 15 consecutive nucleotides of SEQ ID NO:1.

[0014] Furthermore, the dsRNA is prepared using the effect gene Mg-grn-1 of the aforementioned grass root-knot nematode as a template, and it targets and can specifically interfere with or silence the expression of the effect gene Mg-grn-1 in the grass root-knot nematode.

[0015] Preferably, the nucleotide sequence of the dsRNA is shown in SEQ ID NO:30.

[0016] Furthermore, the dsRNA is provided by any of the following materials: A) DNA molecules encoding dsRNA as described in the third aspect; B) Recombinant expression vectors, recombinant gene expression cassettes, recombinant microorganisms, or recombinant cells containing the DNA molecules described in A); C) Preparations made from the recombinant expression vector, recombinant gene expression cassette, recombinant microorganism, or recombinant cell described in B).

[0017] Furthermore, the in vitro RNA interference includes the following steps: (11) Using the polynucleotide described in the first aspect as a template, the dsRNA is prepared by PCR amplification and in vitro transcription; (12) Place the root-knot nematode of the Poaceae family in the solution of dsRNA prepared in step (11) for in vitro treatment.

[0018] Further, in step (11), the plasmid pSuper:Mg-grn-1 containing the effect gene Mg-grn-1 is used as a template, and PCR amplification is performed using specific primers with T7 promoter adapters to obtain a DNA template for in vitro transcription. Then, dsRNA is synthesized using an in vitro transcription kit.

[0019] Furthermore, in step (11), the specific primer sequences with T7 promoter adapters used to amplify the Mg-grn-1 fragment of the root-knot nematode effect gene are shown in SEQ ID NO: 18 and SEQ ID NO: 19.

[0020] Furthermore, in step (11), the dsRNA of the Mg-grn-1 gene fragment is synthesized using the T7 RNAi Transcription Kit and purified using the magnetic bead method.

[0021] Further, in step (12), the pre-infected second-instar larvae (pre-J2) are soaked in 0.5×M9 buffer containing 1.5 μg / μL dsRNA, incubated at 25°C with shaking for 24 hours, and then washed to remove residual dsRNA.

[0022] Furthermore, the construction of transgenic plants resistant to grass-like root-knot nematodes includes the following steps: (21) Constructing an RNAi plant expression vector capable of expressing the polynucleotides described in the first aspect of the present invention; (22) Using Agrobacterium-mediated genetic transformation, the expression vector described in step (21) was introduced into rice embryogenic callus; (23) Screening and regeneration of the transformed tissues were carried out to obtain transgenic rice plants.

[0023] Further, in step (21), the plant expression vector includes a vector for overexpressing the effect gene Mg-grn-1 and a vector for RNAi of the gene.

[0024] Further, in step (21), using the plasmid pSuper:Mg-grn-1 containing the Mg-grn-1 gene sequence as a template, PCR amplification is performed using primers GRN_OE-F (SEQ ID NO: 22) and GRN_OE-R (SEQ ID NO: 23) and primers GRN_RNAi-F (SEQ ID NO: 24) and GRN_RNAi-R (SEQ ID NO: 25) to obtain the fragment GRN_OE for overexpression construction and the fragment GRN_RNAi for RNAi construction.

[0025] Further, in step (21), the GRN_OE fragment and the GRN_RNAi fragment are respectively ligated into the plant expression vectors pNC-Cam3304-35S and pNC-Cam3304-RNAi to construct a recombinant expression vector.

[0026] Further, in step (22), the correctly constructed recombinant expression vector is transferred into Agrobacterium strain EHA105 to obtain the engineered bacteria for transformation.

[0027] Furthermore, in step (22), the Agrobacterium engineered bacteria are used to genetically transform embryogenic callus induced by the mature embryo of the rice variety Kitaake as the recipient material.

[0028] Furthermore, in step (23), the transformed tissues are screened using a culture medium containing Basta herbicide, and transgenic positive plants are confirmed by PCR detection.

[0029] Further, in step (23), RNA is extracted from transgenic positive plants (T0 generation), reverse transcribed into cDNA, and the expression level of the Mg-grn-1 gene is detected by real-time quantitative PCR (qPCR) using rice actin gene OsACT (primer sequences SEQ ID NO: 26 and SEQ ID NO: 27) as an internal reference. Transgenic lines with high expression are screened and their seeds are harvested (T1 generation).

[0030] Furthermore, the plant genome integrates an RNAi expression cassette targeting the Mg-grn-1 gene described in the first aspect of the invention, and exhibits resistance to root-knot nematodes of the Poaceae family.

[0031] A fourth aspect of the present invention is to provide a method for detecting the expression of the Mg-grn-1 gene in the root-knot nematode of the Poaceae family, by using specific primers or probes to detect the Mg-grn-1 gene in the root-knot nematode of the Poaceae family.

[0032] Furthermore, the detection method includes the following steps: (31) Total RNA was extracted from the sample to be tested and reverse transcribed into cDNA; (32) Using primers or probes that can specifically amplify the Mg-grn-1 gene, the cDNA obtained in step (31) was detected by qPCR; (33) Based on the results of the qPCR, determine the relative expression level of the Mg-grn-1 gene.

[0033] Further, in step (31), the sample is selected from any one of the root tissues of infected plants at different time points after inoculation with the root-knot nematode of the Poaceae family.

[0034] Further, in step (32), the sequences of the specific primers are shown in SEQ ID NO: 9 and SEQ ID NO: 10.

[0035] Further, in step (32), the qPCR is standardized using an internal reference gene, which is the actin gene MgACT of the root-knot nematode of the Poaceae family; The primer sequences used to amplify the internal reference gene MgACT are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0036] The fifth aspect of the present invention is to provide the use of a polynucleotide or effector protein in the control of root-knot nematodes of the Poaceae family, wherein the polynucleotide is as described in the first aspect of the present invention, and the effector protein Mg-GRN-1 is as described in any of the second aspects of the present invention.

[0037] Furthermore, the application includes the use of polynucleotides or dsRNAs targeting polynucleotides in the control of root-knot nematodes of the Poaceae family, wherein the polynucleotide is the effector gene Mg-grn-1, and the dsRNA is as described in the third aspect of the present invention.

[0038] Furthermore, the application is selected from at least one of the following applications: application in inhibiting the expression level of the Mg-grn-1 gene of the grass root-knot nematode, application in reducing the infectivity of the grass root-knot nematode, application in preparing an agent that inhibits the infectivity of the grass root-knot nematode, and application in constructing transgenic plants resistant to grass root-knot nematode disease.

[0039] Furthermore, dsRNA was used to prepare a formulation that inhibits infection by root-knot nematodes of the Poaceae family.

[0040] Furthermore, soaking *Poaceae* root-knot nematodes in dsRNA preparations inhibited the expression level of the *Poaceae* root-knot nematode gene Mg-grn-1 and reduced the infectivity of *Poaceae* root-knot nematodes.

[0041] Furthermore, an RNAi plant expression vector containing the effector gene Mg-grn-1 was constructed; the expression vector was introduced into rice embryogenic callus, and the transformed tissues were screened and plants were regenerated to obtain transgenic rice resistant to root-knot nematode disease of the grass family.

[0042] Furthermore, the application is the use of polynucleotide or effector protein Mg-GRN-1 in the preparation of formulations that increase the sensitivity of plants to root-knot nematodes of the Poaceae family, wherein the polynucleotide is the effector gene Mg-grn-1.

[0043] Furthermore, engineered bacteria that express the Mg-grn-1 gene were introduced into the plants to obtain plants with reduced immune responses.

[0044] Furthermore, the reduced immune response is characterized by reactive oxygen species bursts and decreased expression of defense genes.

[0045] Furthermore, using the plasmid pSuper:Mg-grn-1 containing the Mg-grn-1 gene sequence as a template, a recombinant expression vector for expressing the effector protein Mg-GRN-1 was constructed. The correctly constructed recombinant expression vector was then transformed into Agrobacterium strain GV3101 to obtain a preparation that increases the sensitivity of plants to root-knot nematodes of the Poaceae family.

[0046] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: (1) This invention identifies and reveals the function of the key effector gene Mg-grn-1 and its encoded protein Mg-GRN-1 of the root-knot nematode of the Poaceae family. This gene is specifically highly expressed in the early stage of infection. Its encoded effector protein can be localized to the plant cell membrane and cell nucleus, and can significantly inhibit the plant reactive oxygen species burst induced by flg22 and chitin and the expression of defense genes. This invention clarifies the key role of this gene in inhibiting basic immunity during the successful infection of plants by nematodes, and provides a core molecular target for developing new control strategies targeting this effector.

[0047] (2) This invention demonstrates the effectiveness of RNAi technology by synthesizing dsRNA of the Mg-grn-1 gene in vitro and using it to treat nematodes. dsMgGRN1 treatment can significantly reduce the expression level of the Mg-grn-1 gene in the root-knot nematode of the Poaceae family and significantly inhibit the early infection and later colonization ability of nematodes on rice roots. This proves that the pathogenicity of nematodes can be effectively weakened by delivering dsRNA targeting this gene in vitro, laying the application foundation for the development of new biological pesticides based on RNAi formulations.

[0048] (3) This invention successfully constructed Mg-grn-1 overexpressing and RNAi transgenic rice lines, and verified the biological function of this gene at the plant level. Compared with the wild type, the overexpressing lines were more susceptible to nematodes, with a significant increase in the number of nematodes in the roots, the number of root knots, and the reproductive output; while the RNAi-silenced lines showed significantly enhanced resistance, with nematode infection and development severely inhibited. These results clarify that targeting this nematode-effect gene in the plant host through genetic manipulation can effectively improve crop resistance, providing a direct basis and effective technical approach for cultivating new nematode-resistant rice varieties using plant-mediated RNAi or gene editing technology. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the conserved domains, signal peptide, and protein sequence of the Mg-GRN-1 protein of the present invention; in the diagram, A represents the domains of the Mg-GRN-1 protein in SignalP. A) The website 6.0 predicts the Mg-GRN-1 protein signal peptide; B) The website 6.0 predicts the transmembrane structure using HMMTOP; C) The website 6.0 predicts the transmembrane structure using SWISS. After performing three-dimensional structural analysis on MODEL, the Mg-GRN-1 protein sequence and key structural domains were determined.

[0050] Figure 2 The expression levels of the Mg-grn-1 gene in different instars (A) and different infection stages (B) of the root-knot nematode of the Poaceae family are shown in the bar chart. The same letter indicates no significant difference (p>0.05), different uppercase letters indicate extremely significant differences (p<0.01), and different lowercase letters indicate significant differences (p<0.05).

[0051] Figure 3 Subcellular localization of Mg-GRN-1 protein in tobacco leaves.

[0052] Figure 4 The diagram shows the inhibitory effects of Mg-GRN-1 on reactive oxygen species (ROS) bursts (A) and defense gene expression (C) induced by the bacterial flagellin short peptide flg22, and the inhibitory effects of Mg-GRN-1 on chitin-induced ROS bursts (B) and defense gene expression (D).

[0053] Figure 5 The figure shows the silencing efficiency of RNAi on the expression of the Mg-grn-1 gene in second-instar larvae in vitro; in the figure, ns indicates no significant difference, and ** indicates extremely significant difference (p<0.01).

[0054] Figure 6 The effect of in vitro RNAi on the infection efficiency of second-instar larvae on rice is shown in the figure; ns indicates no significant difference, and ** indicates extremely significant difference (p<0.01).

[0055] Figure 7 The expression levels of the Mg-grn-1 gene in different lines of T0 transgenic rice.

[0056] Figure 8 This figure shows the infection and development of root-knot nematodes in transgenic rice with Mg-grn-1 gene overexpression and RNAi T1 generation lines. In the figure, A: nematode staining in rice roots 3 days after inoculation; BC: number of nematodes in rice roots 1 day (B) and 3 days (C) after inoculation; D: nematode staining in rice roots 21 days after inoculation; EF: number of root knots (E) and percentage of nematodes at each age stage (F) in rice roots 21 days after inoculation. In the bar chart, the same letter indicates no significant difference (p>0.05), different uppercase letters indicate extremely significant differences (p<0.01), and different lowercase letters indicate significant differences (p<0.05). Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this application clearer, the application is described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0058] Obviously, the accompanying drawings described below are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0059] In this application, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0060] the term As used herein, an "expression cassette" or "gene expression cassette" refers to a gene expression system containing all the necessary elements required to express a target gene, typically including the following elements: a promoter, a target gene sequence, and a terminator; these elements are operatively linked.

[0061] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0062] As used herein, the term “RNA interference (RNAi)” refers to a mechanism that inhibits the expression of a target gene by introducing a double-stranded RNA (dsRNA) consisting of a strand that is sequence homologous to the mRNA of the target gene and a strand that has a sequence complementary to the aforementioned strand, thereby inducing the degradation of the target gene’s mRNA.

[0063] As used in this article, the gene Mgra_00009828 in the population genome of the root-knot nematode VN-18 of the Poaceae family is abbreviated as Mg-grn-1. The effector protein is Mg-GRN-1.

[0064] As used in this article, "grass root-knot nematode effector protein" is abbreviated as Mg-GRN-1.

[0065] The nematode populations used in the following examples were initially collected from rice paddies in Taizhou City, Jiangsu Province. They were then propagated using single-egg mass culture, as detailed in the literature [Feng Hui, ZHOU C, Zhu Feng, LE Xiu-Hu, JING De-Dao, DalyP, Zhou D, Wei L. Resistance analysis of the rice variety Huaidao 5 against root-knot nematode]. Meloidogyne graminicola . Journal of Integrative Agriculture. 2023, 22(10): 3081-3089].

[0066] The chemical reagents and experimental materials used in the following examples are all commercially available: Test plant: Nicotiana benthamiana ( Nicotiana benthamiana Rice (Kitaake variety) was cultured in pots in an artificial climate chamber (24°C, 16 h light, 6 h dark); rice was cultured in an artificial climate chamber or plant growth box (26°C, 12 h light, 12 h dark). Test strains: Escherichia coli DH5α, Agrobacterium GV3101; Test vectors: pSuper, pSuper:GFP, pNC-Green-SubC, pBI221-mCherry-PM, pCambia1304, pNC-Cam3304-35S, and pNC-Cam3304-RNAi.

[0067] The primers used in the following examples are shown in Table 1 below: Table 1: Primer Sequence Listing Example 1: Obtaining the effect gene Mg-grn-1 of the root-knot nematode (Poaceae family) and its encoded protein sequence Previously, numerous potential effector genes were identified from early-stage second-instar larvae of the grass-like root-knot nematode, encoding proteins with signal peptides but lacking transmembrane structures. To investigate the function of these proteins, some of the effector proteins with unknown functions were transiently co-expressed with the plant immune elicitors INF1 and XEG1 on tobacco leaves, and one effector that could inhibit cell death was screened. Its amino acid sequence includes a granulin-like domain. Based on this, we hypothesize that granular proteins of the grass-like root-knot nematode play a potential role in the nematode's parasitism in plants and could be used as a target for the control of the grass-like root-knot nematode.

[0068] In this embodiment, the sequence of the effect gene Mg-grn-1 and the sequence of its encoded effect protein Mg-GRN-1 of the root-knot nematode of the Poaceae family were obtained through gene cloning and sequence analysis.

[0069] (1) Gene cloning: Total RNA was extracted from the second instar larvae (pre-J2) of the root-knot nematode of the Poaceae family using Trizol reagent (Invitrigen, USA). The RNA was reverse transcribed into cDNA according to the EasyScript one-step gDNA removal and first-strand cDNA synthesis kit (TransGold, Beijing).

[0070] Based on the transcriptome data of the root-knot nematode of the Poaceae family previously completed by our team, one of the effector genes that inhibits cell death induced by the plant elicitor XEG1 was named Mg-grn-1 (Mgra_00009828 gene in the VN-18 population genome of the root-knot nematode of the Poaceae family). A pair of cloning primers, pS-GRN-F and pS-GRN-R, containing Xba I / Sma I restriction sites were designed based on its full-length CDS sequence. The primer sequences are shown in Table 1 as SEQ ID NO: 4 and SEQ ID NO: 6. Using pre-infected second-instar larvae (pre-J2) cDNA as a template, PCR amplification was performed according to the Phanta Max Master Mix (Novazia, Nanjing) reagent instructions. The reaction program was as follows: pre-denaturation 95°C, 3 min; denaturation 95°C, 15 s; annealing 56°C, 15 s; extension 72°C, 15 s, 34 cycles; extension 72°C, 5 min; storage at 4°C for later use.

[0071] Following the instructions of the ClonExpress Ultra One Step Cloning kit (Novizan, Nanjing), the full-length CDS sequence of Mg-grn-1 was introduced into the Xba I / Sma I restriction site of the pSuper vector to construct the recombinant vector pSuper:Mg-grn-1. Sequencing yielded the nucleotide sequence of the Mg-grn-1 gene. The obtained sequence was translated into amino acids, and the protein signal peptide was predicted using the online tool SignalP 6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ). The transmembrane structure was predicted using the online tool TMHMM 2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). SWISS was used to predict the transmembrane structure. Use MODEL (https: / / swissmodel.expasy.org) for 3D structural analysis.

[0072] (2) Sequence analysis like Figure 1 As shown, the open reading frame of the cloned Mg-grn-1 gene is 282 bp in length and has 100% similarity to the published gene sequence of the root-knot nematode Mgra_00009828. The full-length sequence is shown in SEQ ID NO: 1.

[0073] The protein Mg-GRN-1 encoded by the Mg-grn-1 gene consists of 93 amino acids, and its amino acid sequence is shown in SEQ ID NO:2.

[0074] The effector protein Mg-GRN-1 has a signal peptide at its N-terminus. The signal peptide contains 24 amino acids and has no transmembrane domain. Its amino acid sequence is shown in SEQ ID NO: 3.

[0075] The nucleotide sequence encoding the signal peptide is shown in SEQ ID NO: 28.

[0076] The 3D structure of the Mg-GRN-1 protein contains a Granulin-like domain, the sequence of which is shown in SEQ ID NO: 29.

[0077] Example 2: Determination of the expression pattern of the Mg-grn-1 gene in the root-knot nematode of the Poaceae family This embodiment uses qPCR to determine the expression pattern of the Mg-grn-1 gene in the root-knot nematode of the Poaceae family, including the expression pattern in the root-knot nematode of the Poaceae family at different ages, and the expression pattern at different stages of the root tip infection of rice by the root-knot nematode of the Poaceae family.

[0078] (1) Collection of nematodes at different instars: Collect diseased rice root tissues to isolate root-knot nematode eggs and prepare pre-infected second-instar larvae (pre-J2). For other nematode instars, under a stereomicroscope, use a nematode picking needle to pick out infective second-instar larvae (J2), third-instar and fourth-instar larvae (J3 / J4) and female nematodes (FM) from rice root tissues and place them in centrifuge tubes.

[0079] (2) Nematode inoculation and nematode collection at different infection stages: Rice culture and nematode plate inoculation were carried out first. The nematodes in the roots were stained with 0.013% fuchsin solution (containing 0.8% acetic acid) at 12 h, 24 h, 48 h, 72 h and 120 h (hpi) after inoculation to evaluate the nematode infection efficiency.

[0080] (3) Nematodes of various ages and rice root tip tissues at different times after inoculation were frozen in liquid nitrogen, and RNA was extracted using TRIzol reagent (Invitrigen, USA). Following the kit instructions, the extracted RNA was reverse transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Novizan, Nanjing). The actin gene of the root-knot nematode (Poaceae family) was used as an example. MgACTAs internal controls, the primer sequences were qMgACT-F and qMgACT-R, as shown in Table 1 (SEQ ID NO: 7 and SEQ ID NO: 8). The relative expression level of the Mg-grn-1 gene was analyzed according to the procedure and system of the SYBR Green Pro Taq HS Premix kit (Novizan, Nanjing), using primer sequences qGRN-F and qGRN-R, as shown in Table 1 (SEQ ID NO: 9 and SEQ ID NO: 10).

[0081] (4) Experimental results The results are as follows Figure 2 As shown, Mg-grn-1 was mainly expressed in infective second-instar larvae (J2), and its relative expression level was significantly higher than that in eggs, pre-infected second-instar larvae (pre-J2), third and fourth instar larvae (J3 / 4), and female larvae. Figure 2 Part A). Twelve hours after nematode inoculation, the expression level of Mg-grn-1 increased rapidly, reaching its peak at 24 and 48 hours post-inoculation. It began to decline at 72 hours, but remained significantly higher than the expression level of pre-inoculation nematodes (pre-J2). Figure 2 (Part B of the study). The results showed that Mg-grn-1 mainly plays a role in the early infection process of root-knot nematodes.

[0082] Example 3: Subcellular localization of Mg-GRN-1 protein in tobacco leaves In this embodiment, the Mg-grn-1 gene and a transient expression vector of the Mg-grn-1 gene without a signal peptide were constructed, transformed into Agrobacterium (GV3101) and infiltrated into tobacco. The subcellular localization of the Mg-GRN-1 protein in tobacco leaves was obtained by observation using a fluorescence microscope.

[0083] (1) Construction of transient expression vectors: Primers GRNsub-F and GRNsub-R containing the target vector adapter were designed based on the nucleotide sequence of Mg-grn-1. The primer sequences are shown in Table 1, SEQ ID NO: 11 and SEQ ID NO: 13. Simultaneously, primers dGRNsub-F and GRNsub-R containing the target vector adapter were designed based on the nucleotide sequence of Mg-grn-1 without the signal peptide. The primer sequences are shown in Table 1, SEQ ID NO: 12 and SEQ ID NO: 13. Using pSuper:Mg-grn-1 plasmid as a template, PCR amplification was performed using the above primers according to the PCR system and procedure of Example 1. Referring to the instructions of the pNC plant vector construction kit (Hainan Nixing Biotechnology Co., Ltd.), the PCR product from the previous step was ligated into the NC site of the vector pNC-Green-SubC to construct the pNC:Mg-grn-1:GFP vector and pNC: △SP Mg-grn-1:GFP vector.

[0084] (2) Agrobacterium transformation and transient expression: After correct sequencing, Agrobacterium strain GV3101 was transformed and transient expression was performed on tobacco leaves; simultaneously, the cell membrane localization vector pBI221-mCherry-PM was transformed into GV3101 and then fused with pNC:Mg-grn-1:GFP and pNC: △SP Mg-grn-1:GFP was co-expressed on tobacco leaves. The localization of the fusion protein was observed under a confocal microscope (PerkinElmer UltraVIEW VoX, USA) after 2-3 days.

[0085] (3) Experimental results Synthesis of Mg-GRN-1 protein and signal-free peptide using a transient expression system in tobacco leaves △SP Subcellular localization analysis of Mg-GRN-1 protein yielded the following results: Figure 3 As shown, Mg-grn-1 and Mg-grn-1 were injected respectively. △SP Two days after Mg-grn-1 was introduced into Agrobacterium, fluorescent signals were observed on the cell membrane and nucleus of tobacco leaf epidermal cells, indicating that the signal peptide does not affect the localization of Mg-GRN-1 protein, and that Mg-GRN-1 protein is located in the cell membrane and nucleus of plant cells.

[0086] Example 4: Inhibitory effect of Mg-GRN-1 protein on plant basal immunity This embodiment constructs a transient expression vector for the Mg-grn-1 gene and a Mg-grn-1 gene without a signal peptide, transforms Agrobacterium (GV3101) into tobacco, and detects the reactive oxygen species level and the expression of the defense gene CYP71D20 in tobacco to obtain the inhibitory effect of Mg-GRN-1 protein on plant basal immunity, as detailed below: (1) Expression and immune stimulation of Mg-GRN-1 protein: Using pSuper:Mg-grn-1 plasmid as a template, PCR amplification of the protein without the signal peptide sequence was performed using primers pS-dGRN-F and pS-GRN-R. △SP Mg-grn-1, primer sequences are shown in Table 1 as SEQ ID NO: 5 and SEQ ID NO: 6. pSuper was constructed according to the vector construction method described in Example 1. △SP The Mg-grn-1 recombinant vector, containing the signal peptide sequence, is pSuper:Mg-grn-1 as described in Example 1, with the pSuper:GFP vector carrying green fluorescent protein as a negative control. Agrobacterium transformation and transient protein expression were performed using the above vectors following the steps described in Example 3.

[0087] Eighteen hours later, flat tobacco leaves were selected and treated with bacterial flagellin peptide flg22 (amino acid sequence QRLSTGSRINSAKDDAAGLQIA) and chitin as elicitors. Each elicitor treatment experiment was divided into four groups: a GFP+ddH2O group as a blank control, a GFP+ elicitor (flg22 / chitin) group as a negative control, and a Mg-GRN-1+ elicitor group... △SP The Mg-GRN-1+ exciton was used as the experimental group.

[0088] (2) Reactive oxygen species and defense gene CYP71D20 assay: Relative light units (RLU) were measured by chemiluminescence immunoassay using an ELISA reader (BMG Labtech, Germany) to assess reactive oxygen species levels; Total RNA was extracted from the treated tobacco leaves and cDNA was synthesized according to the method in Example 2. (The text then abruptly shifts to a seemingly unrelated topic: using tobacco...) NbACTThe gene was used as an internal control. The primer sequences were qNbACT-F and qNbACT-R, as shown in SEQ ID NO: 14 and SEQ ID NO: 15 in Table 1. The relative expression level of the defense gene CYP71D20 was analyzed according to the procedure and system of the SYBR Green Pro Taq HS Premix kit (Novizan, Nanjing). The primer sequences were qNbCYP71D20-F and qNbCYP71D20-R, as shown in SEQ ID NO: 16 and SEQ ID NO: 17 in Table 1.

[0089] (3) Experimental results: The inhibitory effect of Mg-GRN-1 protein on plant basal immunity was tested using chemiluminescence immunoassay. The results are as follows: Figure 4 As shown. Compared with the blank control group, flg22 and chitin could significantly induce reactive oxygen species bursts in plants, but Mg-GRN-1 and △SP The expression of Mg-GRN-1 protein can inhibit flg22 ( Figure 4 Part A) and chitin ( Figure 4 The B-part of Mg-GRN-1 induces reactive oxygen species (ROS) bursts, and the presence or absence of a signal peptide does not affect the decrease in ROS levels induced by Mg-GRN-1 protein. Meanwhile, Mg-GRN-1 and △SP The expression of Mg-GRN-1 protein can inhibit flg22 ( Figure 4 Part A) and chitin ( Figure 4 The upregulation of the defense gene NbCYP71D20 (part B) leads to the upregulation of Mg-GRN-1 and... △SP Both Mg-GRN-1 protein significantly inhibited flg22 and chitin-induced reactive oxygen species bursts in plants and reduced the expression level of the defense gene NbCYP71D20, indicating that the signal peptide does not affect the biological activity of Mg-GRN-1 protein.

[0090] Example 5: Preparation of Mg-grn-1 gene dsRNA and in vitro RNAi In this embodiment, dsRNA was prepared by in vitro transcription, and the prepared dsRNA was used to perform in vitro RNAi treatment on root-knot nematodes of the Poaceae family, as detailed below: (1) dsRNA preparation: Using pSuper:Mg-grn-1 plasmid as a template, PCR amplification was performed with primers dsGRN-T7F and dsGRN-T7R to obtain the Mg-grn-1 gene fragment with the T7 promoter adapter sequence. The primer sequences are shown in Table 1 as SEQ ID NO: 18 and SEQ ID NO: 19. Simultaneously, using pCambia1304 plasmid as a template, the green fluorescent protein (gfp) gene fragment with the T7 promoter adapter sequence was obtained with primers GFP-T7F and GFP-T7R. The primer sequences are shown in Table 1 as SEQ ID NO: 20 and SEQ ID NO: 21. The dsRNA of the Mg-grn-1 and gfp gene fragments were synthesized according to the instructions of the T7 RNAi Transcription Kit (Novizan, Nanjing) and purified using the magnetic bead method. The nucleotide sequence of Mg-grn-1 dsRNA is shown in SEQ ID NO: 30, and the nucleotide sequence of gfp gene segment dsRNA is shown in SEQ ID NO: 31.

[0091] (2) In vitro RNAi treatment: 1500 pre-J2 nematodes were soaked in 200 μL of 0.5×M9 buffer (containing 1.5 μg / μL dsRNA), incubated at 25°C with shaking for 24 h, and then rinsed with DEPC water to remove residual dsRNA. Nematodes soaked in 0.5×M9 buffer and gfp gene dsRNA solution were used as negative controls. The treated nematodes were collected, and nematode RNA was extracted and cDNA was synthesized according to the methods of Example 1 and Example 2. The expression level of Mg-grn-1 gene was detected. Nematodes were inoculated according to the method of Example 2. Nematode staining was performed and the number of nematodes in the roots was investigated 3 days and 21 days after inoculation.

[0092] (3) RNAi results: such as Figure 5 As shown, compared with 0.5×M9 buffer (M9 buffer) and gfp dsRNA (dsGFP), Mg-grn-1 dsRNA (dsMgGRN1) treatment for 24 h significantly reduced the expression level of Mg-grn-1 in second-instar larvae of root-knot nematodes. Figure 6 As shown, Mg-grn-1 dsRNA treatment significantly inhibited the number of nematodes invading rice roots in the early (3 d) and late (21 d) stages, indicating that interfering with the expression of Mg-grn-1 in vitro can affect the infectivity of root-knot nematodes in rice.

[0093] Example 6: Construction and phenotypic identification of Mg-grn-1 transgenic lines This embodiment constructs a transgenic rice line targeting the Mg-grn-1 gene, and inoculates its progeny plants with the grass-like root-knot nematode to examine the effect of the Mg-grn-1 transgenic line on the reproduction and infection of the grass-like root-knot nematode, as detailed below: Using pSuper:Mg-grn-1 plasmid as a template, the Mg-grn-1 gene fragments GRN_OE and GRN_OE-R were amplified by PCR with primers GRN_OE-F and GRN_OE-R, and GRN_RNAi-F and GRN_RNAi-R, respectively. The primer sequences are shown in Table 1, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. The nucleotide sequence of GRN_OE is shown in SEQ ID NO: 32, and the nucleotide sequence of GRN_RNAi is the nucleotide sequence of Mg-grn-1 dsRNA (as shown in SEQ ID NO: 30).

[0094] Following the instructions of the pNC plant vector construction kit (Hainan Nixing Biotechnology Co., Ltd.), GRN_OE and GRN_RNAi were ligated into the pNC-Cam3304-35S and pNC-Cam3304-RNAi vectors, respectively. After successful sequencing, the vectors were transformed into Agrobacterium strain EHA105. Genetic transformation was performed using callus induced from mature embryos of the rice variety Kitaake via Agrobacterium-mediated transformation. Positive plants were screened using Basta herbicide and PCR detection.

[0095] RNA was extracted from T0 generation rice seedlings and cDNA was synthesized. The rice OsACT gene was used as an internal reference. The primer sequences were qOsACT-F and qOsACT-R, as shown in SEQ ID NO: 26 and SEQ ID NO: 27 in Table 1. Rice lines expressing high Mg-grn-1 were screened by qPCR analysis according to Example 2, and their seeds were harvested.

[0096] T1 generation rice seedlings with high Mg-grn-1 expression were transplanted into plastic pots (9 cm × 9 cm × 12 cm), with 2 seedlings per pot. The pot substrate consisted of equal volumes of sterilized peat moss, paddy soil, and river sand. Inoculation was performed when the rice was at the 4-5 leaf stage. One mL of a suspension containing 20 pre-J2 nematodes was injected into the rhizosphere soil using a pipette. The number of nematodes in the roots was examined under a microscope at 7, 14, and 28 days post-inoculation. Wild rice (Kitaake) was used as a control.

[0097] The relative expression level of the target gene was detected by qPCR, and the results are as follows: Figure 7As shown, among all the lines, the overexpression lines OE#5 and OE#15 and the RNAi lines RNAi#13 and RNAi#19 had the highest expression levels. Therefore, these transgenic lines were used for subsequent resistance and susceptibility analysis. Figure 8 As shown, compared to wild-type rice Kitaake, the number of root nematodes in the overexpression rice lines OE#5 and OE#15 was significantly increased at 1 and 3 days after inoculation with T1 generation transgenic rice seedlings, while the number of root nematodes in the RNAi lines RNAi#13 and RNAi#19 was significantly decreased. Figure 8 Parts A-C). The number of root knots in rice roots 21 days after inoculation was investigated. The results showed that the number of root knots in OE#5 and OE#15 lines was significantly higher than that in Kitaake, while the number of root knots in RNAi#13 and RNAi#19 lines was significantly lower than that in Kitaake. Furthermore, more ovipositing female insects (FML) and eggs were observed in the roots of OE#5 and OE#15 rice, while no ovipositing female insects or eggs were observed in the roots of RNAi#13 and RNAi#19 rice. Figure 8 (Parts D-F). These results indicate that Mg-grn-1 plays an important role in promoting infection and development of the grass root-knot nematode. Overexpression of the effector gene Mg-grn-1 increases plant susceptibility to grass root-knot nematode disease, while silencing the effector gene Mg-grn-1 increases plant resistance to grass root-knot nematodes.

[0098] This invention provides the effector gene Mg-grn-1 of the grass-like root-knot nematode and its encoded effector protein Mg-GRN-1. This protein is located in the plant cell membrane and nucleus and can inhibit the plant's basal immune response. This invention successfully prepared the dsRNA (dsMgGRN1) of the Mg-grn-1 gene through in vitro transcription and successfully obtained Mg-grn-1 overexpressing and RNAi transgenic rice lines through Agrobacterium-mediated transformation. In vitro treatment with dsMgGRN1 significantly reduced the infectivity of the grass-like root-knot nematode; at the plant level, overexpression of Mg-grn-1 increased the susceptibility of rice to nematodes, while silencing the gene enhanced rice resistance to nematodes. This invention provides a new key molecular target for the control of grass-like root-knot nematodes and lays an important foundation for the development of novel targeted control strategies (such as RNAi biopesticides or disease-resistant breeding).

[0099] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A polynucleotide, characterized in that, It contains the nucleotide sequence shown in SEQ ID NO:1, or a variant that has at least 95% sequence identity with SEQ ID NO:1 and encodes a protein with inhibitory plant immune activity.

2. An effector protein, characterized in that, It contains the amino acid sequence shown in SEQ ID NO:2, or a variant that has at least 95% sequence identity with SEQ ID NO:2 and has inhibitory plant immune activity.

3. The effector protein according to claim 2, characterized in that, The first to 24th amino acids at the N-terminus of the effector protein are a signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO:

3.

4. A method for controlling root-knot nematodes of the Poaceae family, characterized in that, In vitro RNA interference was performed on the root-knot nematode of the Poaceae family using double-stranded RNA targeting the polynucleotide of claim 1; and / or, Transgenic plants resistant to root-knot nematodes of the Poaceae family were constructed using double-stranded RNA targeting the polynucleotide of claim 1, wherein the plants include tobacco and rice.

5. The method according to claim 4, characterized in that, The double-stranded RNA is dsRNA, which contains a sequence complementary to at least 15 consecutive nucleotides of SEQ ID NO:

1.

6. The method according to claim 5, characterized in that, The dsRNA is provided from any of the following materials: A) A DNA molecule encoding the dsRNA as described in claim 4; B) Recombinant expression vectors, recombinant gene expression cassettes, recombinant microorganisms, or recombinant cells containing the DNA molecules described in A); C) Preparations made from the recombinant expression vector, recombinant gene expression cassette, recombinant microorganism, or recombinant cell described in B).

7. The method according to claim 5, characterized in that, The in vitro RNA interference includes the following steps: (11) Using SEQ ID NO:1 as a template as described in claim 1, the dsRNA is prepared by PCR amplification and in vitro transcription; (12) Place the root-knot nematode of the Poaceae family in the solution of dsRNA prepared in step (11) for in vitro treatment.

8. The method according to claim 5, characterized in that, The construction of transgenic plants resistant to grass-like root-knot nematodes includes the following steps: (21) Construct a plant expression vector that can express the RNAi target as described in SEQ ID NO:1 as claimed in claim 1; (22) Using Agrobacterium-mediated genetic transformation, the expression vector described in step (21) was introduced into rice embryogenic callus; (23) Screening and regeneration of the transformed tissues were carried out to obtain transgenic rice plants.

9. A method for detecting Mg-grn-1 gene expression in *Poaceae* root-knot nematodes, characterized in that, The Mg-grn-1 gene in the root-knot nematode of the Poaceae family was detected using specific primers or probes.

10. The application of a polynucleotide or effector protein in the control of root-knot nematodes in the Poaceae family, characterized in that, The polynucleotide is as described in claim 1, and the effector protein Mg-GRN-1 is as described in any one of claims 2 to 3.

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