A southern root-knot nematode effector gene, related protein and its application

Abstract

The present invention relates to the field of genetic engineering technology, specifically a southern root-knot nematode effector gene, related protein and its application. The effector gene is Minc3s00173g06739, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The expression of this gene is significantly increased during the nematode infection stage, and the encoded protein is a typical secretory protein, which is localized in the cell membrane and nucleus of tobacco cells. After overexpression, the plant height, petiole length, and root knot diameter of Arabidopsis are significantly greater than those of the wild type. The present invention found that an effector gene is significantly upregulated during the infection process of southern root-knot nematodes, and overexpression significantly affects the plant height and leaf morphology of Arabidopsis and increases susceptibility to southern root-knot nematodes, which is of great significance for analyzing the relevant molecular mechanisms of southern root-knot nematode disease and establishing a comprehensive prevention and control technology strategy for southern root-knot nematode disease in plants.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to an incognita root-knot nematode effector gene, related proteins and applications thereof. Background Art

[0002] The southern root-knot nematode is a common, worldwide plant parasitic nematode, targeting over 3,000 crops, including vegetables, grains, fruit trees, and ornamentals. In the early stages of nematode infection, the plant's photosynthetic rate decreases, root respiration increases at the infection site, biofilm permeability increases, endogenous hormone metabolism is disrupted, nitrogen, phosphorus, and potassium accumulate in the roots, and the content of biomacromolecules such as soluble proteins and reducing sugars increases. Stem growth is inhibited, leaves lack nutrients and turn yellow, and the plant wilts intermittently. Under favorable conditions, repeated root-knot nematode infection can lead to concentrated root knots, forming giant root knots. These knots shorten the host's main root, reduce lateral roots and root hairs, and cause root deformities. Ultimately, root function declines, preventing the plant from properly absorbing water, mineral ions, and fertilizer. At the same time, large amounts of carbohydrates and minerals in the vascular tissue are unloaded by giant cells, resulting in a lack of water and fertilizer necessary for growth and development. The plant exhibits stunted growth, weak stems, yellowing leaves, small, slow-ripening fruits, and ultimately withering and death. Infection by the southern root-knot nematode (S. melanocarpus) can cause yield reductions of 10% to 20% for many crops, and in severe cases, can reach over 75%, or even total crop failure. With the continuous development of protected vegetable horticulture in my country, production areas are expanding, multiple cropping indexes are increasing, and repeated cropping is becoming increasingly prevalent. This has led to an increasing incidence of root-knot nematode damage, posing a serious threat to the development of Chinese agriculture.

[0003] The southern root-knot nematode (Meloidogyne incognita) is a living parasitic nematode that overwinters primarily as eggs in the soil. When environmental conditions are favorable the following year, the infective second-instar larvae (J2) hatch after molting. Stimulated by plant rhizosphere signals, the J2s migrate toward the root apex, penetrate the root elongation zone, and migrate between cells until they reach the root's vascular bundles. There, they induce the development of 4-8 cells into multinucleated giant cells, establishing feeding sites. These giant cells are the sole source of nutrition for the nematode's development and continue to provide nutrients throughout the parasitic process until the nematode completes its life cycle. During the nematode-plant interaction, the nematode produces secretory proteins through its esophageal gland cells (Kyndt et al. 2013). These proteins are secreted into the host plant through the stylet, playing a crucial role in nematode infection, internal migration, parasitism, and the completion of its life cycle.

[0004] Research has shown that during its infection and parasitism stages, the southern root-knot nematode secretes numerous effector factors into host plant cells. These effector factors have multiple functions, including breaking down host cell wall barriers, suppressing host immune responses, inducing and maintaining feeding sites, regulating host cell reactive oxygen species bursts and programmed cell death, and modulating host histone modification and processing mechanisms, transcriptional regulation, and mRNA splicing. Therefore, effector factors are key to the root-knot nematode's successful parasitism and pathogenicity within its host.

[0005] In recent years, with the continued maturity of various detection technologies, research on the functions of nematode effector factors has continued to deepen. In 2003, a library of expressed sequence tags specific to the esophageal gland of the southern root-knot nematode (M. incognita) identified numerous pioneer genes of unknown function, specifically expressed in esophageal gland cells. Proteomic analysis also identified over 500 secreted proteins in pre-parasitic J2 and female adults of the southern root-knot nematode. However, the vast majority of these proteins are pioneer proteins with no known functional domains. Therefore, functional elucidation of these effector proteins is urgently needed, which is of great significance for the development of integrated control strategies for southern root-knot nematode disease in plants. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a southern root-knot nematode effector gene, related proteins and their applications. It was found that the southern root-knot nematode effector gene Minc3s00173g06739 affects the plant photomorphogenesis phenotype by regulating the growth and development of the host, and increases the plant's sensitivity to southern root-knot nematode disease.

[0007] The object of the present invention is achieved through the following technical solution: an incognita root-knot nematode effector gene, the effector gene is Minc3s00173g06739, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] A southern root-knot nematode effector protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] As a preferred technical solution, the effector protein includes a signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.3.

[0010] A primer pair for amplifying the aforementioned incognita root-knot nematode effector gene, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.5.

[0011] The application of the above-mentioned effector gene, the above-mentioned effector protein or the above-mentioned primer pair in increasing the sensitivity of plants to southern root-knot nematode disease.

[0012] As a preferred technical solution, the plant is Arabidopsis thaliana.

[0013] The above-mentioned effector gene, the above-mentioned effector protein or the above-mentioned primer pair is used to influence the growth and development of Arabidopsis thaliana. The effector gene Minc3s00173g06739 increases the plant height of Arabidopsis thaliana and affects the leaf morphology of Arabidopsis thaliana.

[0014] As an optimal technical solution, the growth and development includes leaf shape, plant height, petiole length, and the number of burrs on the leaf epidermis.

[0015] The present invention has the following advantages: The present invention discloses a southern root-knot nematode effector gene, Minc3s00173g06739. The protein encoded by this effector gene has a 20-amino acid signal peptide at the N-terminus. This signal peptide has a secretory function and does not affect the localization of the effector protein. The effector protein is localized to the cell membrane and nucleus in plant cells. After overexpression, the plant height, petiole length, and root knot diameter of Arabidopsis thaliana are significantly greater than those of the wild type. The present invention discovered that an effector gene, Minc3s00173g06739, is significantly upregulated during southern root-knot nematode infection. Overexpression can significantly affect the photomorphogenic phenotype of Arabidopsis thaliana and increase susceptibility to southern root-knot nematodes. This is of great significance for analyzing the relevant molecular mechanisms of southern root-knot nematode disease and establishing a comprehensive technical strategy for the prevention and control of southern root-knot nematode disease in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the conserved protein domains, signal peptide, and protein sequence of the effector protein Minc3s00173g06739 of the present invention; Figure A: Signal peptide prediction of Minc3s00173g06739 on the SignalP-5.0 website; B: Transmembrane structure prediction results using HMMTOP; C: Sequence and key domains of the Minc3s00173g06739 protein after stereostructure analysis using SWISS-MODEL.

[0017] Figure 2 This figure shows the results of secretion detection of Minc3s00173g06739 protein signal peptide using the yeast signal peptide secretion analysis system.

[0018] Figure 3 This is a diagram showing the expression pattern of the effector gene Minc3s00173g06739 of the present invention during the infection process of the southern root-knot nematode.

[0019] Figure 4 This is a diagram showing the transient expression of the recombinant Agrobacterium in Example 1 of the present invention after infiltration injection into tobacco leaves.

[0020] Figure 5The instant expression of the southern root-knot nematode gene Minc3s00173g06739 in Nicotiana benthamiana leaves in Example 1 of the present invention ∆SP Inhibit cell allergic necrosis reaction, where Figure A is the injection sequence diagram, Figure B is the phenotype diagram of tobacco leaves after injection, and Figure C is the statistical diagram of the necrotic area of ​​tobacco leaves.

[0021] Figure 6 The gene Minc3s00173g06739 of the present invention ∆SP Growth phenotype of plants after overexpression in Arabidopsis.

[0022] Figure 7 This is the root disease phenotype of transgenic Arabidopsis thaliana after infection with the southern root-knot nematode. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following:

[0024] Example 1

[0025] 1 Materials and Methods

[0026] 1.1 Experimental Materials

[0027] Test plants: Transgenic Columbia-type Arabidopsis thaliana lines, cultured in pots in a climate chamber, inoculated with 800 second-instar root-knot nematodes (800 J2 / plant) at the rosette stage. Nicotiana benthamiana plants were provided by our laboratory and cultured in pots in a climate chamber (28°C for 16 h of light and 25°C for 8 h of darkness). Test strains: Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 were provided by our laboratory. Yeast strain YTK12 was purchased commercially. Test vectors: pSUC2 and pSUC2-Avr1b were purchased commercially, and the pCAMBIA2300GFP vector was provided by our laboratory.

[0028] 1.2 Experimental methods

[0029] The primer sequences used in this experiment are shown in Table 1:

[0030] Table 1. Primer sequence list

[0031] Primer Name Sequence PCAMBIA2300-Minc3s00173g06739-F GAGAACACGGGGGACTCTAGAATGGCCACCTTTTTCACTTTTACC (SEQ ID NO.4) PCAMBIA2300-Minc3s00173g06739-R TTCTCCCTTACCCATCCCGGGTTTTTCCATATAATTTTTCGCTCCA (SEQ ID NO.5) <![CDATA[pSUC2-Minc3s00173g06739 SP -F]]> CGGAATTTTAATTAAGAATTCATGGCCACCTTTTTCACTTTTACC (SEQ ID NO.6) <![CDATA[pSUC2-Minc3s00173g06739 SP -R]]> CACTATAGGGAGAACCTCGAGTCCCTCAGTTGTGGCAATAATTG (SEQ ID NO.7) <![CDATA[PCAMBIA2300-Minc3s00173g06739 △SP -F]]> GAGAACACGGGGGACTCTAGAATGATGAATACTAATCGAAGTGCTTCA (SEQ ID NO.8) <![CDATA[PCAMBIA2300-Minc3s00173g06739 △SP -R]]> TTCTCCCTTACCCATCCCGGGTTTTTCCATATAATTTTTCGCTCCA (SEQ ID NO.9) MiACT-RT-F AAGACGAAGCAGCTGTAGCC (SEQ ID NO.10) MiACT-RT-R GGTGTTACGCACACAGTTCC (SEQ ID NO.11) <![CDATA[Minc3s00173g06739 -RT-F ]]> AATTGTATCAGGTGGCAACTCA (SEQ ID NO.12) Minc3s00173g06739-RT-R CATTCTACAGGGTTTCCGTCAT (SEQ ID NO.13) <![CDATA[PGR107-Minc3s00173g06739 △SP -F]]> TCAGCACCAGCTAGCATCGATATGATGAATACTAATCGAAGTGCTTCA (SEQ ID NO.14) <![CDATA[PGR107-Minc3s00173g06739 △SP -R]]> GTGGTCCTTGTAGTCCCCGGGTTTTTCCATATAATTTTTCGCTCCA (SEQ ID NO.15) <![CDATA[PD- Minc3s00173g06739 △SP -F]]> GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGATGAATACTAATCGAAGTGC (SEQ ID NO.16) <![CDATA[PD- Minc3s00173g06739 △SP -R]]> GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATTTTTCCATATAATTTTTCGCT (SEQ ID NO.17)

[0032] 1.2.1 Gene cloning and sequence analysis

[0033] Total RNA from the second larvae (J2) of the incognita root-knot nematode (Meloidogyne incognita) was extracted using the Trizol method and reverse-transcribed into cDNA using the reverse transcription system and protocol of the Revert Aid FirstStrand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, USA). The full-length CDS sequence of the Minc3s00173g06739 gene from the M. incognita V3 genome database (https: / / parasite.wormbase.org / Meloidogyne_incognita_prjeb8714 / Info / Index / ) was used to design a partial cloning primer, PCAMBIA2300-Minc3s00173g06739-F and PCAMBIA2300-Minc3s00173g06739-R, containing XbaI / SmaI restriction sites. The primer sequences are shown in SEQ ID NOs. 4 and 5 in Table 1. PCR amplification was performed using J2 cDNA as a template using the following amplification system:

[0034] cDNA (200 ng / μL) template 2 μL, primers (10 μM) 0.8 μL each, ddH2O 6.4 μL, PremeSTARMax Ver. 2 Premix 10 μL. PCR program: 94°C 3 min, 30 x (94°C 30 s, 55°C 30 s, 72°C 40 s), 72°C 10 min, storage at 4°C.

[0035] The full-length CDS sequence of the Minc3s00173g06739 gene was introduced into the XbaI / SmaI sites of the pCAMBIA2300 vector using the ClonExpress-Entry One-Step Cloning kit (Vazyme, China) to construct the pCAMBIA2300:Minc3s00173g06739:GFP vector. The nucleotide sequence of the Minc3s00173g06739 gene was obtained after sequencing. The obtained sequence was translated into amino acids and used for signal peptide prediction using SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ). Transmembrane structure prediction was performed using HMMTOP (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). Three-dimensional structure analysis was performed using SWISS-MODEL (https: / / swissmodel.expasy.org).

[0036] 1.2.2 Verification of signal peptide secretion function

[0037] Using pCAMBIA2300: Minc3s00173g06739: GFP plasmid as template, a cloning primer containing EcoRI / XhoI restriction sites was designed based on the signal peptide analysis results of Minc3s00173g06739. SP -F and pSUC2 Minc3s00173g06739 SP -R amplify the signal peptide nucleotide sequence of Minc3s00173g06739, the primer sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 in Table 1, the procedure and system are the same as 1.2.1, the nucleotide sequence of the signal peptide of Minc3s00173g06739 is ligated into the EcoRI / XhoI site of the vector pSUC2 to construct pSUC2:Minc3s00173g06739 SPAfter sequencing, the recombinant vector was transformed into the sucrase-deficient yeast YTK12 using the lithium acetate method. Positive transformants were screened and cultured on CMD-W medium (tryptophan-deficient). Positive transformants and YTK12 were diluted in 0.9% NaCl, and 3 μL was spotted on YPRAA medium. The growth of circular spots was observed and compared with the positive control (transformant containing pSUC2-Avr1b) and the negative control (YTK12). The positive transformants and control yeast were also tested for sucrase activity in sucrose solution to verify their secretion function.

[0038] 1.2.3 Analysis of the expression pattern of the Minc3s00173g06739 gene

[0039] Diseased tomato roots were collected and disinfected with 0.5% NaOCl for 10 min, then rinsed several times with distilled water. Root-knot nematode eggs were collected according to the method of Liu Weizhi (2000) and incubated at 28°C for 24 h to obtain J2s according to the method of Fudali et al. (2013). The collected J2s were resuspended in distilled water and the concentration was adjusted to 1000 J2 mL -1 The suspension was inoculated when the cucumber seedlings had two leaves and one heart. The roots of the cucumber seedlings were collected 5, 15, and 30 days after inoculation, and the roots were cut into 0.3 cm root segments after cleaning. They were digested with 6% cellulase (Cellulase, 9012-54-8, Yuanye, China). After sieving, the parasitic J2, third-instar larvae / fourth-instar larvae (J3 / J4), and female worms were collected into centrifuge tubes and stored in a -80ºC refrigerator. The hatched J2 was treated in the same way and used as a control. Total RNA from nematodes was extracted using the Trizol method, and cDNA was synthesized according to the instructions of the reverse transcription kit (Revert Aid First Strand cDNA Synthesis Kit, Roche Diagnostics, Indianapolis, USA). The southern root-knot nematode gene was used. MiACT The primer sequences were MiACT-RT-F and MiACT-RT-R, as shown in SEQ ID NO. 10 and SEQ ID NO. 11 in Table 1, and the relative expression of the Minc3s00173g06739 gene was analyzed with reference to the procedure and system in the instruction manual of Taq Pro Universal SYBR QPCR Master Mix (Vazyme, China). The primer sequences were Minc3s00173g06739-RT-F and Minc3s00173g06739-RT-R, as shown in SEQ ID NO. 12 and SEQ ID NO. 13 in Table 1.

[0040] 1.2.4 Subcellular localization analysis

[0041] Using the pCAMBIA2300: Minc3s00173g06739: GFP plasmid as a template, a cloning primer, PCAMBIA2300-Minc3s00173g06739, containing an XbaI / SmaI restriction site was designed based on the nucleotide sequence of Minc3s00173g06739 without a signal peptide. △SP -F and PCAMBIA2300-Minc3s00173g06739 △SP -R, the primer sequences are shown in SEQ ID NO.8 and SEQ ID NO.9 in Table 1, the procedure and system are the same as 1.2.1, and the PCR product is ligated into the XbaΙ / SmaΙ site of the vector PCAMBIA2300 with reference to the method of the ClonExpress-Entry One Step Cloning kit (Vazyme, China), to construct PCAMBIA2300: Minc3s00173g06739 △SP After correct sequencing, the pCAMBIA2300: Minc3s00173g06739: GFP vector and the pCAMBIA2300: Minc3s00173g06739: GFP vector were respectively transformed into Agrobacterium tumefaciens strain GV3101 for transient transformation of tobacco. The localization of the fusion protein was observed under a confocal microscope (LEICA TCS SP8, Germany) 2-3 days later.

[0042] 1.2.5 Allergic cellular necrosis

[0043] Using the pCAMBIA2300: Minc3s00173g06739: GFP plasmid as a template, the one-step cloning primer PGR107-Minc3s00173g06739 containing the ClaI / SmaI restriction site was designed based on the nucleotide sequence of Minc3s00173g06739 without a signal peptide. △SP -F and PGR107-Minc3s00173g06739 △SP -R, the primer sequences are shown in SEQ ID NO.14 and SEQ ID NO.15 in Table 1, the procedures and system methods are the same as 1.2.1, the PCR product is connected to the ClaI / SmaI site of the vector PGR107 to construct PGR107: Minc3s00173g06739 △SP:GFP vector, after correct sequencing, was transformed into Agrobacterium tumefaciens GV3101 strain and activated overnight with shaking. The cells were harvested by centrifugation and resuspended in buffer, adjusting the absorbance of the suspension to OD600 = 0.5. The buffer formulation was: 10 mM MgCl2, 10 mM MES, 200 μM acetosyringone. Tobacco plants cultured in the greenhouse for 4-5 weeks were selected and the transfection site carrying the effector gene PGR107: Minc3s00173g06739 was injected into the culture medium using a syringe with a needle removed. △SP 200 µL of Agrobacterium carrying pGR107:GFP was injected between cells of the lower epidermis of tobacco leaves. pGR107:GFP and buffer were used as negative and blank controls, respectively. 24 hours later, Agrobacterium carrying pGR107:Bax was injected. Five days after injection, tobacco leaves were observed for necrosis at each injection site, and the percentage of necrotic area at the injection site relative to the total injected area was calculated (0-100%).

[0044] 1.2.6 Phenotypic identification of transgenic Arabidopsis

[0045] Vernalized Arabidopsis thaliana seeds were seeded directly in a 3:1:1 mixture of peat:perlite:vermiculite and cultured in an artificial climate chamber under conditions of 5000 LX light intensity, 95% humidity, 22 / 24°C day and night, and 12 h of light duration per day. The plant expression vector pB2HA: Minc3s00173g06739 was constructed using Gateway. ∆SP , according to Minc3s00173g06739 ∆SP Sequence design of BP reaction primer PD-Minc3s00173g06739 △SP -F and PD-Minc3s00173g06739 △SP -R, the sequence is shown in SEQ ID NO.16 and SEQ ID NO.17 in Table 1, and the intermediate expression vector pDONR201: Minc3s00173g06739 was constructed by replacement reaction. ∆SP Then, the plant expression vector pB2HA: Minc3s00173g06739 was constructed through LR reaction. ∆SP After sequencing was correct, the plasmid was transformed into Agrobacterium GV3101. ∆SPTransform wild-type Arabidopsis thaliana with Agrobacterium tumefaciens containing the plasmid GV3101. Harvest T0 seeds and sow them in MS medium supplemented with 3.5 mg / L Basta. Transplant the surviving strains to the above-mentioned incubator for culture and harvest T1 seeds. Further spray one thousandth of Basta for screening, and obtain T2 generation transgenic homozygous strains through resistance screening, PCR detection and offspring segregation ratio statistics. Wild-type Arabidopsis thaliana and transgenic strains were planted using the sand culture method, and the seedlings were irrigated once a day with 1 / 2 Hoagland nutrient solution. Prepare J2 suspension according to the method in 1.2.3 and adjust the concentration to 200 J2 mL -1 At the rosette stage, 1 mL of the suspension was inoculated per plant by root irrigation, with wild-type Arabidopsis as the control. The number of root nodes was counted 15 days after inoculation, and the growth and development phenotype of Arabidopsis was observed.

[0046] 2. Results Analysis

[0047] 2.1 Sequence analysis

[0048] like Figure 1As shown, the cloned ORF of the Minc3s00173g06739 gene is 555 bp long and has a 95.6% similarity to the published incognita gene sequence. The full-length sequence is: (SEQ ID NO. 1)

[0049] Encoding 184 amino acids (aa), the amino acid sequence is: MATFFTFTLLIISIIATTEGMNTNRSASTSDSLKDQKDCKVIYGMFVPVAGSKMHGDAKSAMKPNNPSLPNKLIVSGGNSKYSVTLQVENQPKCVAQNDGNPVECQIQGDKLSGKLIYDIENGPSVNVPFKDTPIFVGNKCEIVFVAYDKDHKLTLLMNKVKLMIEPTNKQIVKACGAKNYMEK (SEQ ID NO. 2)

[0050] There is a 20 aa signal peptide at the N-terminus, and the signal peptide sequence is: MATFFTFTLLIISIIATTEG (SEQ ID NO. 3),

[0051] The nucleotide sequence of the signal peptide is: ATGGCCACCTTTTTCACTTTTACCCTTCTAATCATTTCAATTATTGCCACAACTGAGGGA (SEQ ID NO. 18), without a transmembrane domain. The three-dimensional structure of the Minc3s00173g06739 protein contains a similar viral transcription regulatory element VP1, the sequence of which is: IYDIENGPSVNVPFKDTPIFVGNKCEIVFVAYDKDHKLTLLMNKVKL (SEQ ID NO. 19).

[0052] 2.2 The signal peptide of Minc3s00173g06739 protein has secretory function

[0053] like Figure 2 As shown, the positive control YTK12[pSUC2-Avr1b] can grow on YPRAA medium, while the negative control YTK12[pSUC2] cannot grow on YPRAA plates; the experimental group YTK12[pSUC2-Minc3s00173g06739 SP ] can grow on YPPAA; the positive control YTK12[pSUC2-Avr1b] secretes sucrase, which can hydrolyze sucrose into monosaccharides. Monosaccharides react with TTC to produce red, water-insoluble triphenyltetrazolium chloride. The negative control YTK12[pSUC2] does not secrete sucrase and cannot produce a significant color reaction with TTC. The experimental group YTK12[pSUC2-Minc3s00173g06739 SP ] secreted sucrase, and the results were consistent with those of the positive control, indicating that the signal peptide of Minc3s00173g06739 has secretory function.

[0054] 2.3 Induced expression of the Minc3s00173g06739 gene during infection with the southern root-knot nematode

[0055] RNA of the southern root-knot nematode was extracted from J2 and at different time points during the infection process, and reverse transcribed into cDNA. The expression level in J2 before infection was used as a control (relative expression level was 1) to analyze the expression pattern of the Minc3s00173g06739 gene. Figure 3 As shown, the results showed that the expression of Minc3s00173g06739 gene was significantly upregulated after the nematode invaded the root system, reaching the highest peak when the nematode developed into the J3 / J4 stage. Although the expression of Minc3s00173g06739 gene decreased significantly after developing into female nematodes, it was still significantly higher than the expression level in J2 before infection.

[0056] 2.4 Localization analysis of the fusion protein expressed by the Minc3s00173g06739 gene in tobacco cells

[0057] The Minc3s00173g06739 protein and Minc3s00173g06739 without signal peptide were expressed in tobacco leaves transiently. ∆SP Subcellular localization analysis of the binding protein. Figure 4 As shown, the results showed that injection of Minc3s00173g06739 and Minc3s00173g06739 ∆SP Two days after infection with Agrobacterium, fluorescent signals could be observed on the cell membrane and nucleus of the lower epidermal cells of tobacco leaves, indicating that the signal peptide did not affect the localization of the Minc3s00173g06739 protein and that the Minc3s00173g06739 protein was localized on the cell membrane and nucleus in plant cells.

[0058] Five days later, GFP was injected into tobacco leaves, and 24 hours later, Bax was injected. The tobacco leaves showed obvious cell necrosis phenotype. After the injection of effector factors and Bax, the average proportion of cell necrosis area in the tobacco leaves decreased by 50%, indicating that the effector factors play an important role in inhibiting the allergic necrosis reaction caused by BAX.

[0059] 2.5 Growth phenotype analysis of transgenic Arabidopsis lines

[0060] like Figure 5 As shown, compared with wild-type Arabidopsis, Minc3s00173g06739 ∆SP Arabidopsis thaliana transformation was performed, and three T2 generation homozygous lines were obtained, named OE1, OE2, and OE3. Their leaf shape and plant height changed significantly, with petioles significantly growing, leaf epidermal burrs reduced, and plant height significantly increased.

[0061] 2.6 Analysis of resistance of transgenic Arabidopsis lines to southern root-knot nematode disease

[0062] like Figure 6 As shown, 15 days after inoculation with southern root-knot nematodes, Minc3s00173g06739 ∆SP The root knots of the transgenic lines were concentrated near the main root, and the root knots were significantly larger than those of the control. Therefore, the Minc3s00173g06739 gene plays an important role in increasing the susceptibility to southern root-knot nematodes.

[0063] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A method for increasing the sensitivity of Arabidopsis thaliana to southern root-knot nematode disease, characterized in that: Upregulating the expression of an effector gene or effector protein, the effector gene is Minc3s00173g06739, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the effector protein is shown in SEQ ID NO.

2.

2. The method for increasing the sensitivity of Arabidopsis thaliana to southern root-knot nematode disease according to claim 1, characterized in that: The effector protein includes a signal peptide, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.

3.

3. The method for increasing the sensitivity of Arabidopsis thaliana to southern root-knot nematode disease according to claim 1, characterized in that: The nucleotide sequence of the forward primer for amplifying the effector gene Minc3s00173g06739 is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

5.

4. A method for increasing the plant height of Arabidopsis thaliana and affecting the leaf morphology of Arabidopsis thaliana, characterized in that: Increasing the expression of the effector gene or increasing the expression of the effector protein, the effector gene is Minc3s00173g06739, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the effector protein is shown in SEQ ID NO.

2.

5. The method for increasing the plant height of Arabidopsis thaliana and affecting the leaf morphology of Arabidopsis thaliana according to claim 4, characterized in that: The leaf morphology includes leaf shape, petiole length and the number of burrs on the leaf surface.

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