The TaCaMBP-like gene and its encoded protein for wheat Phillips cyst nematode infection and its application

Abstract

This invention relates to the wheat susceptibility gene TaCaMBP-like, its encoded protein, and its applications. The amino acid sequence of the wheat susceptibility TaCaMBP-like protein is shown in SEQ ID NO: 2, and the gene sequence encoding this protein is shown in SEQ ID NO: 1. The TaCaMBP-like gene provided by this invention is specifically expressed in wheat roots. Analysis of the TaCaMBP-like gene function using VIGS technology revealed that silencing this gene significantly affects the pathogenicity of the nematode, with a significantly lower number of white female susceptible nematodes in wheat roots compared to the control. The experimental results confirm that this gene plays an important role in the parasitism of susceptible nematodes and can serve as a target gene for nematode resistance engineering. This invention is of great significance for disease resistance breeding research and control of susceptible nematodes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a TaCaMBP-like gene derived from common cultivated wheat, its encoded protein, and its applications. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of my country's important staple crops, with an annual planting area of ​​approximately 2.333 × 10⁻⁶. 7 hm 2 Annual output exceeds 1.38 × 10 8 China's wheat production accounts for approximately 17.5% of global wheat production (National Bureau of Statistics www.stats.gov.cn), and its safe production plays a crucial strategic role in ensuring national food security (Dong Ye, Jian Jinzhuo, Peng Deliang, et al. Research progress on the mechanism of wheat cyst nematode disease and control technology in my country [J]. Acta Phytopathologica Sinica, 2025, 55(4):832-845.). According to statistics from the Food and Agriculture Organization of the United Nations (FAO) in 2019 (http: / / www.fao.org / faostat / zh / #search / wheat), my country's wheat consumption accounts for more than 16% of the world's wheat consumption, and wheat plays a pivotal role in my country's food structure. In recent years, my country's wheat production has repeatedly reached new highs. From 2016 to 2020, my country's wheat production was consistently around 1.33 × 10⁻⁶. 8 The total supply of wheat is basically sufficient (Jiang Yun, Zhang Lili, Xue Ping, et al. Development of my country's wheat industry and international experience reference [J]. China Agricultural Science and Technology Guide, 2021, 23(7):1-10.). However, its production is seriously threatened by a variety of diseases all year round, which can cause huge yield losses and significantly reduce grain quality (Chen Wanquan. Integrated pest management system for major wheat diseases and pests [J]. Plant Protection, 2013, 39(5):16-24.).

[0003] Wheat cyst nematode disease is an important soil-borne disease caused by pathogens such as Heterodera avenae and H. filipjevi. It has occurred and caused damage in more than 40 countries and regions around the world (Dong Ye, Jian Jinzhuo, Peng Deliang, et al. Research progress on the disaster mechanism and control technology of wheat cyst nematode disease in my country [J]. Acta Phytopathologica Sinica, 2025, 55(4):832-845.). Although the distribution of *Heterodera filipjevi* is more widespread in my country than that of *Heterodera filipjevi*, the latter is more virulent and has a longer infection cycle on wheat roots. It not only directly causes more serious damage but also induces various soil-borne fungal diseases. Therefore, *Heterodera filipjevi* poses a more severe overall threat to wheat. (Ren Haohao, Chen Kunyuan, Zhou Bo, et al. Occurrence, distribution and species identification of cyst nematodes in cereal crops in Henan Province [J]. Journal of Plant Protection, 2022, 49(6): 1685-1696; Sahin E, Nicol JM, Elekcioglu L, et al. Hatching of *Heterodera filipjevi* in controlled and natural temperature conditions in Turkey [J]. Nematology, 2010, 12(2): 193-200; Lü Yan, Chen Kunyuan, Lu Junfeng, et al. Investigation on pre-winter infection and damage of wheat cyst nematodes in Xuchang, Henan [J]. China Plant Protection Guide, 2022, 42(12): 38-42+54).

[0004] Wheat cyst nematode disease is a highly damaging soil-borne disease. Due to the scarcity of resistant germplasm resources for wheat cyst nematodes, no wheat varieties have yet been identified that are completely immune to the disease (Kong Ling'an, Cui Jiangkuan, Peng Huan, et al. Research progress on wheat resistance to wheat cyst nematodes [J]. Acta Phytopathologica Sinica, 2021, 51(4): 487-495). Current research on wheat cyst nematode disease mainly focuses on the cereal cyst nematode, while research on the function of resistance-related genes in *Spirogyrus spp.* is relatively limited. Therefore, identifying stable and effective resistance-related genes is of great significance for the control of wheat *Spirogyrus spp.* disease and provides a theoretical basis for the discovery of resistance genes and resistance breeding. Summary of the Invention

[0005] This invention provides a TaCaMBP-like protein derived from wheat, which is specifically expressed in wheat roots. Experiments show that this gene is associated with resistance to *Spirogyrus filamentosa* and can be used as a target gene for plant nematode resistance.

[0006] The TaCaMBP-like protein of wheat susceptible to Phillips cyst nematode disease has the amino acid sequence shown in SEQ ID NO: 2.

[0007] Genes encoding TaCaMBP-like proteins.

[0008] The gene encoding the TaCaMBP-like protein is named TaCaMBP-like, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0009] Application of genes encoding TaCaMBP-like proteins in negatively regulating plant resistance to Phillips cyst nematode disease.

[0010] The application involves reducing the infection, parasitism, development, and / or reproduction of plants by TaCaMBP-like genes by inhibiting or reducing their expression levels or inhibiting the function of their encoded proteins, thereby mitigating the damage caused by TaCaMBP-like nematodes to plants.

[0011] In this application, the virus-induced gene silencing technology VIGS is used to silence the gene encoding the TaCaMBP-like protein, thereby reducing the expression level of the TaCaMBP-like gene and inhibiting the infection and development of Filippodium sp. in plants, thus achieving the purpose of controlling Filippodium sp.

[0012] The primers used in the VIGS method are:

[0013] VCaMBP-like-1F: 5'- CCTTAATTAA CTAAGCAGCCTGTGATGGCAT-3',

[0014] VCaMBP-like-1R: 5'- TATGCGGCCGC TTATTCCGCTTTTCTGCTTTG-3';

[0015] or

[0016] VCaMBP-like-2F: 5'- CCTTAATTAA ACGCCCTCAGGCGTCATCATG-3',

[0017] VCaMBP-like-2R: 5'- TATGCGGCCGC CTTGCTTCTGTTGACGTCATC-3'.

[0018] The *Philippine cyst nematode* refers to the second, third, and fourth instar stages of *Philippine cyst nematode*.

[0019] The plant is wheat and / or barley.

[0020] This invention discovered a TaCaMBP-like protein in wheat, the sequence of which is shown in SEQ ID NO: 2, and the gene sequence encoding this protein is shown in SEQ ID NO: 1. Compared with related sequences in existing publicly available databases, the TaCaMBP-like protein of this invention shows a high sequence similarity to the uncharacterized protein LOC119299607 in *Triticum dicoccoides*, but this homologous protein is only a predicted protein, and its function has not been disclosed. Experiments show that the expression level of this TaCaMBP-like protein is higher in the roots of wheat infected with *Sp. Phillips*, indicating that this TaCaMBP-like protein is a protein associated with wheat susceptibility to *Sp. Phillips*. This invention is the first to demonstrate, through VIGS silencing and mutant materials, that inhibiting TaCaMBP-like gene expression can reduce *Sp. Phillips* infection and the formation of white female nematodes, clarifying the use of this gene as an engineering target for wheat nematode resistance.

[0021] The present invention relates to the application of the TaCaMBP-like protein in inhibiting the parasitism and damage of *Spirometra filamentosa* to plants, and / or inhibiting the pathogenicity of *Spirometra filamentosa* to plants, and / or inhibiting the development of *Spirometra filamentosa*. The substance inhibiting TaCaMBP-like gene expression can specifically be a viral vector that inhibits TaCaMBP-like expression. The host plant can specifically be wheat and barley, such as wheat variety 'Aikang 58' and barley variety Morex.

[0022] This invention utilizes VIGS technology to silence the TaCaMBP-like gene. After inoculation with *Spirometra spp.* for 7 days, the number of second-instar larvae in the roots was significantly reduced compared to the water control group. A 50-day survey of white female nematodes on wheat roots showed that the number of white female nematodes produced by the silenced plants was significantly lower than that of the control. Multiple experiments have confirmed that the TaCaMBP-like gene is associated with the pathogenicity of *Spirometra spp.* and plays an important role, making it a potential target gene for plant nematode resistance engineering. This invention has significant research value for understanding the pathogenic mechanism and control of *Spirometra spp.*

[0023] This invention is the first to clearly demonstrate that the TaCaMBP-like gene negatively regulates resistance to wheat Fischg cyst nematode disease. Inhibiting the expression of this gene can effectively reduce the parasitism and development of Fischg cyst nematode in wheat roots, and has a clear new use and application value. Attached Figure Description

[0024] Figure 1 For the detection of TaCaMBP-like gene PCR amplification;

[0025] Figure 2 Analysis of TaCaMBP-like gene expression induced by *Nematodeus philippinensis*;

[0026] Figure 3 The effect of VIGS-mediated TaCaMBP-like silencing on wheat nematode resistance;

[0027] Figure A: Relative expression level of TaCaMBP-like gene after VIGS silencing; B: Effect of different treatments on nematode infectivity 7 days after inoculation; C: Effect of different treatments on the number of white female nematodes 50 days after inoculation. Data in the figures are mean ± standard error.

[0028] Figure 4 The effects of wild-type (WT) Jimai 22 and Jimai 22 mutant materials on the infection and white female formation of Filippos cyst nematode;

[0029] Figure A: Number of nematodes in the roots of wild-type (WT) and mutant Jimai 22 materials 7 days after inoculation with *Philippines cyst nematode*; Figure B: Number of nematodes in the roots of wild-type (WT) and mutant Jimai 22 materials 14 days after inoculation with *Philippines cyst nematode*; Figure C: Number of white female nematodes in the roots of wild-type (WT) and mutant Jimai 22 materials 50 days after inoculation with *Philippines cyst nematode*. Data in the figures are mean ± standard error.

[0030] Figure 5 The effects of Kronos wild-type (WT) and Kronos mutant materials on the infection and white female formation of *Nematodeus Phillips*.

[0031] Figure A: Number of nematodes in the roots of Kronos wild-type (WT) and Kronos mutant materials 7 days after inoculation with *Nematodeus spp.*; Figure B: Number of nematodes in the roots of Kronos wild-type (WT) and Kronos mutant materials 14 days after inoculation with *Nematodeus spp.*; Figure C: Number of white female nematodes in the roots of Kronos wild-type (WT) and Kronos mutant materials 50 days after inoculation with *Nematodeus spp.*. Data in the figures are mean ± standard error. Detailed Implementation

[0032] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are commercially available. All experimental materials used by the applicant are publicly available. The wheat materials Aikang 58, Jimai 22, and barley Morex used in this invention are all publicly available materials; the Kronos mutant material is derived from a publicly available mutant resource library or publicly available mutant materials; the VIGS vector and related plasmid materials are preserved in the applicant's laboratory. The Fischer cyst nematode population is derived from a publicly available field-collected population, which is preserved in the applicant's laboratory. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0033] Example 1

[0034] Cloning of the TaCaMBP-like gene includes the following steps:

[0035] 1. Take a 2 cm long wheat leaf and put it into a 2 mL RNase-free centrifuge tube. Immediately extract total RNA from wheat using the Trizol method and reverse transcribe it into cDNA using the SuperScript™ IV First-Strand Synthesis System kit.

[0036] 2. Using cDNA as a template, PCR amplification was performed using the upstream primer TaCaMBP-like-F: 5'-ATGACCGAGGAGGTGATCAAAC-3' and the downstream primer TaCaMBP-like-R: 5'-GGAAATGCTGGGTCTGCCTTGC-3'. The amplification system was: KOD One™ PCR Master Mix, 25 µL; forward primer (10 mM), reverse primer (10 mM), 1 µL each; cDNA first-strand template, 4 µL; ddH2O, 19 µL, total volume 50 µL. The amplification program was: 98℃ pre-denaturation for 5 min; followed by 35 cycles of 98℃ for 10 s, 60℃ for 5 s, and 68℃ for 15 s extension; a final extension at 72℃ for 5 min, and storage at 4℃. 5 μL of the PCR product was analyzed by agarose gel electrophoresis (see [link to product details]). Figure 1 The amplified fragment size is 1833 bp.

[0037] 3. DNA was recovered using an agarose gel DNA recovery kit, then ligated into the T vector and transformed into *E. coli* DH5α competent cells. Positive colonies were picked and sequenced (see SEQ ID NO: 1). Sequencing results showed that the amplified product contained the open reading frame shown in SEQ ID NO: 1 of the sequence listing, encoding the protein shown in SEQ ID NO: 2 of the sequence listing. Sequence 2 of the sequence listing was named TaCaMBP-like protein, and its encoding gene was named TaCaMBP-like gene.

[0038] Example 2

[0039] Analysis of TaCaMBP-like gene expression induced by *Nematodeus sp. Phillips* includes the following steps:

[0040] 1. Place a large number of sporangia of Phillips cysts on a 100-mesh screen, spread them evenly in a 9 cm glass dish, add an appropriate amount of sterile water, and incubate in the dark at 16℃. After 3 days, collect the second instar larvae (J2) from the hatching pool and prepare a suspension of 500 larvae / mL for later use.

[0041] 2. The susceptible wheat variety *Aikang 58* was selected for germination. When the wheat roots reached 2-3 cm in length, 100 mL of sterilized sand (Vsand:Vsoil = 1:3) was added to a 15 cm high, 3 cm diameter PVC tube. One wheat seed was sown in each tube, and 1 mL of nematode suspension (500 nematodes / mL) was added to the root. The surface was covered with 2 cm of sand and incubated in a light incubator (day:night = 16 h:8 h) at 18 / 14℃. Root, stem, and leaf samples were collected at 3, 5, 7, 10, and 15 days after inoculation, with roots, stems, and leaves from uninoculated wheat serving as controls. Wheat roots were rinsed with sterile water, blotted dry on sterilized filter paper, frozen in liquid nitrogen, and immediately stored at -80℃. Each treatment was replicated three times.

[0042] 3. Total RNA from wheat was extracted using the Trizol method, and cDNA was obtained by reverse transcription using the SuperScript™ IV First-Strand Synthesis System kit.

[0043] 4. Real-time quantitative PCR was used to detect the expression level of wheat TaCaMBP-like genes. Based on the cDNA sequence of the wheat TaCaMBP-like gene and the wheat internal reference gene (β-actin gene), specific quantitative PCR primers were designed using Primer Premier 5.0 software. The qRT-PCR primer sequences were: RT-CaMBP-like-F: 5'-CAAGTATCCTGCCAGACGCA-3' and RT-CaMBP-like-R: 5'-CGCGGTCTCCTTGATCACAT-3'; β-actin-F: 5'-GAAGTGCTTTTGAAGAGTCGGT-3' and β-actin-R: 5'-CCTGTACCCCTTATTCCTCTGAG-3'. Real-time quantitative PCR reaction system: 2×SuperReal PreMix Plus, 10 µL; forward primer (10 mM), reverse primer (10 mM), 0.6 µL each; cDNA, 1 µL; 50×ROX Reference Dye, 0.4 µL; RNase-free ddH2O, 7.4 µL, total volume 20 µL. Reaction program: pre-denaturation at 50℃ for 2 min; 95℃ for 15 min; denaturation at 95℃ for 10 s; extension at 60℃ for 32 s, for a total of 40 cycles; fluorescence signal acquisition at 95℃ for 10 s; 60℃ for 1 min; 95℃ for 1 s. Application 2 -△△Ct The relative gene expression level was calculated using the method described in Derveaux et al., 2010. The formula is: ΔCT = CT 目的基因 -CT 内参基因 , ΔΔCT=ΔCT 样品 -ΔCT 对照 .

[0044] 5. The results of qRT-PCR are as follows: Figure 2 As shown, the expression level of the TaCaMBP-like gene was upregulated by 2.4-fold in the roots at 15 days, and was also upregulated in the leaves and stems. These results indicate that the TaCaMBP-like gene is induced by Fischg phlebotomyces.

[0045] Example 3

[0046] The TaCaMBP-like gene was silenced using VIGS technology to verify its role as a target in the infection and development of *Nematodeus Phillips*.

[0047] 1. Primers were designed based on the cDNA sequence of the TaCaMBP-like gene using Primer Premier 5.0 software. PacI and NotI restriction sites and protective bases were added to the 5' end of the primers. The forward primer was prefixed with CCTTAAATTAA, and the reverse primer with TATGCGGCCGC. The VIGS primer sequence is as follows:

[0048] VCaMBP-like-1F: 5'- CCTTAATTAA CTAAGCAGCCTGTGATGGCAT-3',

[0049] VCaMBP-like-1R: 5'- TATGCGGCCGC TTATTCCGCTTTTCTGCTTTG-3';

[0050] VCaMBP-like-2F: 5'- CCTTAATTAA ACGCCCTCAGGCGTCATCATG-3',

[0051] VCaMBP-like-2R: 5'- TATGCGGCCGC CTTGCTTCTGTTGACGTCATC-3'.

[0052] 2. Construction of the recombinant vector

[0053] The target fragment of the TaCaMBP-like gene was amplified using the specific primers described above. The DNA fragment was recovered using a column PCR product purification kit, ligated into a vector, transformed into DH5α, and sequenced. The plasmid carrying the target gene and the BSMV:γ-PDS vector (preserved in the applicant's laboratory and available for distribution) were double-digested with PacI and NotI, respectively. After incubation at 37°C for 3 hours, the gene-specific fragment and the γ-vector fragment were recovered by 1.5% agarose gel electrophoresis and ligated using T4 DNA Ligase. The ligation product was transformed into DH5α, and positive clones were screened by colony PCR. The plasmid was extracted and sequenced using primer γ-seq-F: 5´-AAAGTGAGGTTAACGCAATACG -3´ to complete the construction of the recombinant γ-vector (BSMV:γ-TaCaMBP-like-1 / 2).

[0054] Among them, BSMV:γ0 is the empty vector control, and BSMV:γ-PDS is the positive control vector, used to observe the whitening phenotype of wheat leaves after PDS gene silencing, in order to verify whether the BSMV-VIGS system is effective; BSMV:γ-TaCaMBP-like-1 and BSMV:γ-TaCaMBP-like-2 are the recombinant γ vectors used to silence the TaCaMBP-like gene in this embodiment.

[0055] 3. Carrier linearization

[0056] Viral vectors α, β, γ0, BSMV:γ-PDS, and recombinant γ plasmids were extracted. α and BSMV:γ0 plasmids were digested with MluI; β plasmid with SpeI; and BSMV:γ-PDS and recombinant γ plasmids were digested with BssHII. After incubation at 37℃ for 2 h, 0.5 μL of the solution was diluted and electrophoresed to confirm complete linearization. The enzymes were then inactivated by incubation at 65℃ for 15 min.

[0057] 4. In vitro transcription

[0058] Using RiboMAX TM The Large Scale RNA Production Systems-T7 was used to perform in vitro transcription of the linearized plasmid. The specific reaction system is described in the instruction manual. The above reaction mixture was added to a microcentrifuge tube and incubated at 37°C for 2 h. 0.5 μL of the in vitro transcription product was diluted 10-fold with DEPC-treated water for electrophoresis detection and stored at -80°C for later use.

[0059] 5. Take 2.5 μL each of α, β, and BSMV:γ0, BSMV:γ-PDS, BSMV:γ-TaCaMBP-like-1, and BSMV:γ-TaCaMBP-like-2 in vitro transcription products, mix them in a 1:1:1 ratio, and dilute with an equal volume of DEPC-treated water. Take 2.5 μL of the mixture and add 45 μL of FES buffer, and mix thoroughly using a micropipette. Take 8-10 μL of the mixture and rub the second leaf of the seedling three times to complete the inoculation. After inoculation, spray a small amount of DEPC-treated water, maintain room temperature at 23 ± 2˚C, keep moist and dark for 24 h, and then carry out normal 16 / 8 h light / dark cycle culture. Each independent experiment includes a negative control inoculated with FES buffer, a blank control of BSMV:γ0, and a positive control of BSMV:γ-PDS. Observe the viral infection symptoms and PDS phenotype regularly.

[0060] This embodiment includes the following treatments: WT control, i.e., wheat plants that were not inoculated with the virus or were only inoculated with FES buffer; BSMV:γ0 empty vector control, i.e., wheat plants inoculated with α, β and BSMV:γ0 in vitro transcription products; BSMV:γ-PDS positive control, i.e., wheat plants inoculated with α, β and BSMV:γ-PDS in vitro transcription products, used to observe the albino phenotype produced by PDS silencing; and BSMV silencing treatment, i.e., wheat plants inoculated with α, β and BSMV:γ-TaCaMBP-like-1 or BSMV:γ-TaCaMBP-like-2 in vitro transcription products, respectively.

[0061] 6. Ten days after wheat inoculation with the virus, BSMV:γ-PDS plants exhibited a distinct albino phenotype. Samples were taken to detect the relative expression level of the TaCaMBP-like gene. Wheat plants in each treatment were then inoculated with nematodes. 1 mL of nematode suspension (500 nematodes / mL) was added to the wheat roots, covered with 2 cm of sand, and incubated in a light incubator (day:night = 16 h:8 h) at 18 / 14℃ for 7 days, 20 / 16℃ for 7 days, 25 / 18℃ for 21 days, and 30 / 22℃ for 7 days. Seven days after inoculation, three wheat plants from each treatment were stained with acid fuchsin, and the number of nematodes in the roots was counted under a microscope. Fifty days later, white female nematodes were collected using a simple flotation method and counted under a microscope.

[0062] 7. The results showed that, compared with WT and BSMV:γ0, the expression levels of TaCaMBP-like genes in silenced plants were significantly decreased. Figure 3 (A) Seven days after inoculation, acid fuchsin staining was performed on wheat roots of each treatment to investigate the early nematode infection status. The results showed that the nematode infection levels in the BSMV:γ-TaCaMBP-like-1 and BSMV:γ-TaCaMBP-like-2 treatments were significantly lower than those in the BSMV:γ0 empty vector control, decreasing by 69.49% and 69.46%, respectively. Figure 3 (B). After 50 days, an investigation of white female insects in each treatment revealed that the treatment significantly reduced the number of white female insects forming at the wheat roots by 89.47% and 78.95% ( ). Figure 3 (C)

[0063] The above results indicate that silencing the TaCaMBP-like gene significantly reduces the number of early-stage Fischus cysts infecting wheat roots and inhibits the formation of later-stage white females. Therefore, TaCaMBP-like is a susceptibility-related gene in wheat closely associated with Fischus cyst infection and development, and inhibiting its expression can improve wheat resistance to Fischus cyst disease.

[0064] Example 4

[0065] The role of the TaCaMBP-like gene in the infection and development of *Nematodeus Phillips* was verified using the hexaploid wheat Jimai 22 mutant and the tetraploid wheat Kronos mutant.

[0066] 1. The candidate genes were compared in the Jimai 22 gene mutation library (http: / / 39.98.48.156:8822 / # / ) to find the Jimai 22 mutant materials that could produce premature termination or amino acid variation for each gene. Among them, the mutant system corresponding to the TaCaMBP-like gene was the termination variant material. Phenotypic identification was performed on them, with wild-type Jimai 22 (WT) plants as controls.

[0067] Simultaneously, a mutant material corresponding to the TaCaMBP-like gene in the Kronos background was selected for parallel validation. This Kronos mutant material formed a premature stop codon at the corresponding site of the TaCaMBP-like gene, leading to premature termination of gene expression. Kronos wild-type (WT) plants were used as a control. Phenotypic identification of the Jimai 22 mutant material and the Kronos mutant material, representing two genetic backgrounds, was used to jointly verify the role of the TaCaMBP-like gene in the infection and development of *Nematodeus philippinii*.

[0068] 2. Select the above-mentioned wheat varieties for germination. When the wheat roots are 2-3 cm long, add 100 mL of sterilized sand (V sand:V soil = 1:3) into a PVC pipe with a height of 15 cm and a diameter of 3 cm. Sow one wheat seed in each pipe and add 1 mL of nematode suspension (500 nematodes / mL) to the roots. Cover the surface with 2 cm of sand and then incubate in a light incubator (day:night = 16 h:8 h) at 18 / 14℃.

[0069] 3. Seven and fourteen days after inoculation, three wheat plants from each treatment were taken and stained with acid fuchsin. The number of root nematodes was counted under a microscope. After 50 days, white female nematodes were collected using a simple flotation method and counted under a microscope.

[0070] 4. The results showed that there was no significant difference in nematode infection levels between the Jimai 22 mutant and the wild-type Jimai 22 plants at 7 days. Figure 4 (A) There was no significant difference in nematode infection levels among the Jimai 22 mutant plants at 14 days ( Figure 4 (B) The number of white female insects formed at 50 days decreased significantly. Figure 4 (C), a decrease of 81.63%.

[0071] Compared with wild-type Kronos plants, there was no significant difference in nematode infection levels in the Kronos mutant at 7 days. Figure 5(A) There was no significant difference in nematode infection levels in Kronos mutant plants at 14 days. Figure 5 (B) The number of white female insects formed at 50 days decreased significantly. Figure 5 The concentration of C (in the middle) decreased by 68.75%, indicating a significant increase in the plant's disease resistance.

[0072] This demonstrates that TaCaMBP-like genes negatively regulate wheat resistance to Phillips cyst nematode and play an important role in the development of Phillips cyst nematode. Inhibiting the expression of this gene can effectively prevent Phillips cyst nematode from damaging wheat.

Claims

1. The TaCaMBP-like protein of wheat susceptible to Phillips cyst nematode disease, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The gene encoding the TaCaMBP-like protein of claim 1.

3. The gene according to claim 2 is named TaCaMBP-like, and its nucleotide sequence is shown in SEQ ID NO:

1.

4. The application of the gene encoding TaCaMBP-like protein as described in claim 2 or 3 in the negative regulation of plant resistance to Phillips cyst nematode disease.

5. The application according to claim 4, which aims to reduce the infection, parasitism, development and / or reproduction of plants by *Philippia filamentosa* by inhibiting or reducing the expression level of TaCaMBP-like genes or inhibiting the function of their encoded proteins, thereby mitigating the damage caused by *Philippia filamentosa* to plants.

6. The application according to claim 5 is to silence the gene encoding TaCaMBP-like protein using VIGS technology, thereby reducing the expression level of TaCaMBP-like protein, which leads to the inhibition of infection and development of plants by Filippodium sp., thereby achieving the purpose of controlling Filippodium sp.

7. In the application according to claim 6, the primers used in the VIGS technology are: VCaMBP-like-1F:5’- CCTTAATTAA CTAAGCAGCCTGTGATGGCAT-3’, VCaMBP-like-1R:5’- TATGCGGCCGC TTATTCCGCTTTTCTGCTTTG-3’; or VCaMBP-like-2F:5’- CCTTAATTAA ACGCCCTCAGGCGTCATCATG-3’, VCaMBP-like-2R:5’- TATGCGGCCGC CTTGCTTCTGTTGACGTCATC-3’。 8. The application according to any one of claims 4-7, wherein the Phillips cyst nematode is in the second, third, or fourth instar stage of the Phillips cyst nematode.

9. The application according to claim 8, wherein the plant is wheat and / or barley.

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