Cnaphalocrocis medinalis sialoprotein CmFer1, coding gene and application of Cnaphalocrocis medinalis sialoprotein CmFer1 in improving insect resistance of plants

Abstract

The invention provides a cnaphalocrocis medinalis salivary protein CmFer1, a coding gene and application of the cnaphalocrocis medinalis salivary protein CmFer1 in improving insect resistance of plants, and belongs to the technical field of genetic engineering disease control. It is found for the first time that the coding gene of the cnaphalocrocis medinalis elicitor CmFer1 protein can activate a plant immune system and improve the insect resistance of plants, a new thought for activating a plant defense system through insect elicitors is developed, guiding of breeding of rice insect-resistant varieties is facilitated, and a new thought is provided for green control of cnaphalocrocis medinalis. A gene overexpression technology proves that the expression of the CmFer1 in the tobacco can obviously enhance the resistance of the tobacco to the spodoptera frugiperda, a new functional gene and a new technical path are provided for insect-resistant molecular breeding of crops, the use of chemical pesticides is expected to be reduced, and the agricultural production cost and the environmental risk are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering disease prevention and control, and particularly relates to a rice leaf folder salivary protein CmFer1, an encoding gene and an application thereof in improving plant insect resistance. Background Art

[0002] Over the course of evolution, plants and insects have developed an offensive and defensive "arms race" relationship. Insect oral secretions (OS), a key component of plant-insect interactions, play a crucial role in regulating defense responses. Insect OS contain two distinct types of proteins: effectors and elicitors. Insects secrete effectors to suppress plant defense responses, while plants recognize insect elicitors and trigger immune responses to defend against insect attacks.

[0003] The rice leaf folder (Cnaphalocrocis medinalis), a member of the Lepidoptera order and Crambidae family, is a significant migratory pest of rice. During feeding, the larvae bind rice leaves lengthwise into husks, where they feed on the epidermis and mesophyll, ultimately causing white rice leaves. While the saliva components of the rice leaf folder's OS have been identified, the key elicitor remains unknown. Summary of the Invention

[0004] The present invention aims to provide a rice leaf folder salivary protein CmFer1, an encoding gene and an application thereof in improving plant insect resistance, which can effectively improve plant insect resistance.

[0005] The present invention provides a rice leaf folder salivary protein CmFer1, wherein the rice leaf folder salivary protein CmFer1 comprises any one of the following:

[0006] 1) A protein comprising the amino acid sequence shown in SEQ ID NO. 1;

[0007] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having an identity of more than 80% with the protein described in 1) and having the effect of improving plant insect resistance;

[0008] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0009] The present invention also provides a homologous protein of the rice leaf folder salivary protein CmFer1 described in the above scheme, characterized in that it comprises at least one of the proteins with amino acid sequences shown in SEQ ID NOs. 2 to 5.

[0010] The present invention also provides a gene CmFer1 encoding the rice leaf folder salivary protein CmFer1 described in the above scheme.

[0011] Preferably, the coding gene CmFer1 includes any one of the following:

[0012] (1) A gene comprising the nucleotide sequence shown in SEQ ID NO. 6;

[0013] (2) A gene encoding the rice leaf roller salivary protein CmFer1 according to claim 1, obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1);

[0014] (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

[0015] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0016] The present invention also provides a recombinant bacterium comprising the recombinant vector described in the above scheme.

[0017] The present invention also provides the use of the rice leaf folder salivary protein CmFer1, the homologous protein, the encoding gene CmFer1, the recombinant vector or the recombinant bacteria in improving plant insect resistance or breeding insect-resistant plants.

[0018] Preferably, the plant includes tobacco and / or rice; the improving the insect resistance of the plant includes improving the resistance of the plant to pests; and the pest is Spodoptera frugiperda.

[0019] The present invention also provides a method for improving plant insect resistance, comprising the following steps: overexpressing the rice leaf folder salivary protein CmFer1, the homologous protein or the encoding gene CmFer1 in the above scheme in plants.

[0020] The present invention also provides a method for subcellular localization of the coding gene CmFer1 in plants, comprising the following steps: transiently co-expressing the coding gene CmFer1 with a plasma membrane localization marker gene, and observing the subcellular localization of the CmFer1 protein.

[0021] The present invention provides a rice leaf roller salivary protein CmFer1, wherein the rice leaf roller salivary protein CmFer1 comprises any of the following: 1) a protein comprising the amino acid sequence shown in SEQ ID NO. 1; 2) a protein obtained by replacing, deleting, and / or adding one or more amino acid residues of the protein in 1) and having more than 80% identity with the protein shown in 1) and having the effect of improving plant insect resistance; 3) a fusion protein obtained by linking the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag. The present invention is the first to discover that the gene encoding the rice leaf roller elicitor CmFer1 protein can activate the plant immune system and provide insect resistance to the plant, opening up a new idea for using insect elicitors to activate the plant defense system, helping to guide the selection and breeding of insect-resistant rice varieties and providing a new idea for the green control of rice leaf rollers. Gene overexpression technology has confirmed that the expression of CmFer1 in tobacco can significantly enhance tobacco's resistance to the fall armyworm, providing new functional genes and technical paths for the molecular breeding of insect-resistant crops, which is expected to reduce the use of chemical pesticides and reduce agricultural production costs and environmental risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The salivary protein CmFer1 induces tobacco cell necrosis and immune response; A is the cell necrosis symptom on Nicotiana benthamiana leaves, CmFer1 is the full-length ferritin, GFP is the negative control, INF1 is the positive control, CmFer1 ΔSP A is ferritin without signal peptide; B is the detection of reactive oxygen species (ROS) of defense substances; C is the accumulation of salicylic acid (SA) related genes NPR1 and PR1a; D is the accumulation of jasmonic acid (JA) related genes LOXD and AOS; E is the content of SA in tobacco leaves after transient expression; F is the content of JA in tobacco leaves after transient expression;

[0024] Figure 2 is the expression pattern of the salivary gene CmFer1; A is the expression pattern of CmFer1 in different tissues; B is the expression pattern of CmFer1 in different developmental stages;

[0025] Figure 3Subcellular localization of CmFer1; CmFer1 and PM (plasma membrane marker) were transiently co-expressed in Nicotiana benthamiana leaves; control GFP and RFP (cytoplasm and cell membrane co-localization marker) were transiently co-expressed in Nicotiana benthamiana leaves;

[0026] Figure 4 The salivary protein CmFer1 induces tobacco insect resistance; A is the feeding preference of Spodoptera frugiperda on tobacco leaves transiently expressing GFP and CmFer1; B is the number of Spodoptera frugiperda on tobacco leaves transiently expressing GFP and CmFer1,

[0027] Figure 5 This is a functional conservation analysis of insect Fer1 proteins; A is a phylogenetic analysis of Fer1 proteins; B is the functional conservation of herbivorous insect Fer1 proteins in inducing tobacco cell necrosis. DETAILED DESCRIPTION

[0028] The present invention provides a rice leaf folder salivary protein CmFer1, wherein the rice leaf folder salivary protein CmFer1 comprises any one of the following:

[0029] 1) A protein comprising the amino acid sequence shown in SEQ ID NO. 1;

[0030] 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having an identity of more than 80% with the protein described in 1) and having the effect of improving plant insect resistance;

[0031] 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

[0032] The present invention also provides a gene CmFer1 encoding the rice leaf folder salivary protein CmFer1 described in the above scheme.

[0033] The rice leaf folder salivary protein CmFer1 of the invention is an elicitor-ferritin in the rice leaf folder OS, and the elicitor can induce necrosis of tobacco leaf cells and generate immune response.

[0034] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is specifically:

[0035] MKMMLFALAALLAVCAPAYATQCHVNPVNIPKEWITMHRPCRDSMRRQIQMEVAASLQYLAMGAHFSKDTVNRPGFAKLFFEAGSEEREHAMKLIEYMLMRGELTS DVTSLITIRPPERKTWGNGVEALEHALRMESDVTKSIRTVITACEDDSEFNDYHLVDYLTGEFLEEQYKGQRDLAGKASTLKKMLDRHPALGEFIFDKKLMGIDV*.

[0036] The present invention also provides a homologous protein of the rice leaf folder salivary protein CmFer1 described in the above scheme, including at least one of the proteins with amino acid sequences shown in SEQ ID NOs. 2 to 5.

[0037] In the present invention, the protein with the amino acid sequence shown in SEQ ID NO.2 is SfFer1, specifically:

[0038] MKAILFALAGLLAVCLPASATQCHVNPVTIPKEWVTMHRNCRMSMRQQIQMEVAASLQYLAMGAHFSKDIVNRPGFAKMFFDAASEEREHAMKLIEYLLMRGELTT DVTTLLQVRAPERKTWEGGVEALEHALLMESAVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLDEQYKGQRDIAGKASTLKKLLDRHGALGEFIFDKKLIGIDI*.

[0039] In the present invention, the protein with the amino acid sequence shown in SEQ ID NO. 3 is NIFer1, specifically: MSSVITFLLAFCMILLVECQNANDRCSIDMDDTLEKVEWKTMHSNCTLEVKDQIKMEYNAAMIYLSLGVHFSRDFVNRPGFAKFFFESASEERQHAIKLIEYLSMRGESVTDIAKLVKLDPETMPGMASVSLNGKEALEKALQQEVLVTNNILKVMKACENEEVKDAAWTLPNDYHLVDWLTAEFLDEQYKGQRDIAGKLSTLLKMGSSNYHLGEFLFDKKLLSNEA*.

[0040] In the present invention, the protein with the amino acid sequence shown in SEQ ID NO.4 is SeFer1, specifically: MKAILLALAGLLAVCLPASATQCHVNPVTIPKEWVSMHRNCRMSMRQQIQMEVTASLQYLAMGAHFSKDVVNRPGFAKMFFDAASEEREHAMKLIEYLLMRGELTSDVTSLLQVRAPERKTWEGGVEALEHALLMESAVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDIAGKASTLKKLLDRHGALGEFIFDKKLIGIDI*

[0041] In the present invention, the protein with the amino acid sequence shown in SEQ ID NO.5 is HaFer1, specifically: MKAILLAVVGLLAVCLPASATQCHVNPVNIPKEWVTMVRNCRMSMRQQIQMEVAASLQYLAMGAHFSRDVVNRPGFAQMFFDAASEEREHAMKLIEYLLMRGELTSDVTTLLQVRAPERKTWEGGVEALEHALRMESEVTKSIRNVIKSCEDDSEFNDYHLVDYLTGDFLEEQYKGQRDIAGKASTLKKLMDRHGALGEFIFDKKLIGIDI*.

[0042] As an embodiment, the coding gene CmFer1 includes any one of the following:

[0043] (1) A gene comprising the nucleotide sequence shown in SEQ ID NO. 6;

[0044] (2) A gene encoding the rice leaf roller salivary protein CmFer1 according to claim 1, obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1);

[0045] (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

[0046] In the present invention, the nucleotide sequence shown in SEQ ID NO.6 is specifically:

[0047] .

[0048] In the present invention, the coding gene CmFer1 is highly expressed in the intestine (foregut and midgut) of the rice leaf roller larvae; the intestine includes the foregut and midgut; and the coding gene CmFer1 is highly expressed in the adult stage of the rice leaf roller at different developmental stages.

[0049] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0050] In one embodiment, the recombinant vector is a plant overexpression vector; the backbone plasmid of the plant overexpression vector is pBIN-GFP, which contains a GFP fluorescent tag. In one embodiment, the method for constructing the plant overexpression vector includes: constructing the coding gene CmFer1 into pBIN-GFP to obtain the plant overexpression vector pBIN-GFP-CmFer1.

[0051] As an embodiment, the present invention also provides a primer pair for constructing the encoding gene CmFer1 into pBIN-GFP; the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.7, specifically: "GACGAGCTGTACAAGGGTACCATGAAGATGATGCTGTTTG; the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO.8, specifically: GCGGACTCTAGTTCATCTAGATTAGACGTCGATGCCCATG.

[0052] The present invention also provides a recombinant bacterium comprising the recombinant vector described in the above scheme.

[0053] As an embodiment, the recombinant bacteria is recombinant Agrobacterium; the original bacteria of the recombinant Agrobacterium are Agrobacterium GV3101 competent cells; the construction method of the recombinant Agrobacterium includes: transforming the recombinant vector into Agrobacterium GV3101 competent cells, expanding the culture after successful transformation, obtaining the bacteria by centrifugation, and resuspending the bacteria in a buffer solution.

[0054] As an embodiment, the OD600 of the bacterial solution of the recombinant bacteria is adjusted to 0.4.

[0055] The present invention also provides the use of the rice leaf folder salivary protein CmFer1, the homologous protein, the encoding gene CmFer1, the recombinant vector or the recombinant bacteria in improving plant insect resistance or breeding insect-resistant plants.

[0056] As an embodiment, the plant includes tobacco and / or rice; the tobacco includes Nicotiana benthamiana.

[0057] As an embodiment, the improving the insect resistance of plants includes improving the resistance of plants to pests; the pests are fall armyworms; and the fall armyworms are second-instar larvae of fall armyworms.

[0058] The present invention also provides a method for improving plant insect resistance, comprising the following steps: overexpressing the rice leaf folder salivary protein CmFer1 or the encoding gene CmFer1 in the above scheme in a plant.

[0059] As an embodiment, the method for overexpressing the rice leaf folder salivary protein CmFer1 or the encoding gene CmFer1 in plants comprises: infecting plant leaves with the recombinant bacteria described in the above scheme; the infection method comprises injecting a bacterial suspension of the recombinant bacteria.

[0060] Overexpressing the coding gene CmFer1 in tobacco can effectively improve tobacco's resistance to the fall armyworm.

[0061] The present invention also provides a method for subcellular localization of the coding gene CmFer1 in plants, comprising the following steps: transiently co-expressing the coding gene CmFer1 with a plasma membrane localization marker gene, and observing the subcellular localization of the CmFer1 protein.

[0062] To further illustrate the present invention, the following describes in detail the rice leaf folder salivary protein CmFer1, the encoding gene and its application in improving plant insect resistance provided by the present invention in conjunction with the drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Acquisition and Sequence Analysis of the CmFer1 Gene: The applicant previously identified 229 salivary proteins in the rice leaf roller (OS) (Cuietal., 2024). The candidate protein coding sequences screened through bioinformatics analysis were constructed into the plant transient expression vector pBIN-GFP using a homologous recombination kit (NoviZan, Cat. No. C112-02). Agrobacterium was then transformed and injected into tobacco, ultimately yielding the salivary protein CmFer1, which can induce necrosis in Nicotiana benthamiana cells. The amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding the salivary protein CmFer1 is shown in SEQ ID NO: 2.

[0065] The CmFer1 gene was constructed into pBIN-GFP digested with KpnI and XbaI to obtain the plant overexpression vector pBIN-GFP-CmFer1. The upstream and downstream primers for constructing the CmFer1 gene into the plant expression vector pBIN-GFP are shown in SEQ ID NO. 4 for the upstream primer and SEQ ID NO. 5 for the downstream primer.

[0066] The pBIN-GFP-CmFer1 recombinant plasmid was transformed into Agrobacterium GV3101 competent cells by chemical transformation to obtain Agrobacterium containing the plasmid.

[0067] Example 2

[0068] Effects of transient expression of CmFer1 on resistance to Nicotiana benthamiana

[0069] The recombinant Agrobacterium containing CmFer1 obtained in Example 1 was inoculated into 5 mL of LB liquid medium and cultured at 28°C and 220 rpm. After 24 hours, the culture was collected and suspended in recombinant Agrobacterium washing buffer (1 M MgCl2, 500 mM MES, 100 μM acetosyringone). The OD600 of the culture was adjusted to 0.4 and injected into approximately 4-week-old Nicotiana benthamiana leaves.

[0070] 1. Symptoms of cell necrosis

[0071] Three days after injection, the necrotic symptoms of tobacco leaves were observed. Figure 1 As shown in A: The positive control INF1 (oomycete elicitor) can induce tobacco cell necrosis (Chen et al., 2023). After expressing the CmFer1 gene containing the signal peptide, the tobacco leaves also showed cell necrosis symptoms. △sp ) and green fluorescent protein (GFP) genes, there was no obvious change in tobacco leaves.

[0072] 2. Determination of Reactive Oxygen Species (ROS) Accumulation

[0073] 24 hours after injection, the leaves were collected and completely immersed in diaminobenzidine (DAB) solution and incubated at room temperature in the dark for 8 hours. The DAB staining solution was then removed and the excess staining solution on the leaves was washed off with pure water. The leaves were then incubated with anhydrous ethanol at 65°C until they completely lost their green color. Finally, the leaves were transferred to 20% glycerol to rehydrate them. The brown color on the leaves is ROS. Figure 1 As shown in Figure B: Similar to the positive controls INF1 and TePDI (tetranychus edulis elicitor) (Cui et al., 2023), transient expression of the CmFer1 gene can induce ROS accumulation in tobacco, while GFP cannot induce ROS accumulation.

[0074] 3. Determination of salicylic acid (SA) and jasmonic acid (JA) content

[0075] Detection of the expression levels of SA and JA related genes: 24 hours after injection, leaves expressing CmFer1 and GFP genes were collected, total RNA was extracted and reverse transcribed. The reverse transcribed cDNA was diluted 10 times and used for quantitative PCR to detect the expression levels of SA related genes NPR1 and PR1a, as well as the expression levels of JA related genes LOXD and AOS. NbRPL23 was selected as the internal reference gene of Nicotiana benthamiana. The following reaction was performed on an ABI 7500 Real-Time PCR instrument: pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 10s, extension at 60℃ for 30s, and denaturation and extension cycles for 35 times. The differential expression fold of genes between different samples was calculated by 2 -ΔΔCT The results are as follows. Figure 1 As shown in Figures C and D, compared with transient expression of GFP, the CmFer1 gene can significantly induce the expression of SA-related gene NPR1, JA-related gene LOXD and AOS gene in Nicotiana benthamiana leaves.

[0076] Detection of SA and JA content in leaves: 24 hours after injection, leaves expressing CmFer1 and GFP genes were collected and the content of tobacco hormones (JA and SA) in different treatment groups and time points was quantitatively analyzed by LC-MS (liquid chromatography-mass spectrometry). Figure 1 As shown in Figures E and F, the SA and JA contents in the leaves of Nicotiana benthamiana transiently expressing the CmFer1 gene were significantly higher than those in the GFP expression group.

[0077] Example 3

[0078] Analysis of the expression pattern of CmFer1

[0079] Samples from different developmental stages (eggs, first-instar larvae, second-instar larvae, third-instar larvae, fourth-instar larvae, fifth-instar larvae, pupae, female adults, and male adults) and different tissues of fourth-instar larvae (salivary glands, foregut, midgut, hindgut, Malpighian tubules, and fat bodies) were collected, total RNA was extracted, and cDNA was reverse transcribed. The cDNA was diluted 10 times and then subjected to quantitative PCR detection. The RPL18 gene was used as the internal reference gene for the rice leaf roller, and the nucleotide sequences of the quantitative primers for the CmFer1 gene are shown in SEQ ID NO.9 (CATCCGCACAGTCATCAC) and SEQ ID NO.10 (GGTCGAGCATCTTCTTGAG). The results are shown in Figure 2. Figure 2 As shown in Figure 2, CmFer1 gene is highly expressed in the intestine (foregut and midgut) of rice leaf roller larvae in different tissues ( Figure 2 A in the figure); At different developmental stages, the CmFer1 gene is highly expressed in the adult stage of Cnaphalocrocis medinalis ( Figure 2 B) in.

[0080] Example 4

[0081] Subcellular localization of CmFer1 in Nicotiana benthamiana

[0082] Bacteria expressing GFP empty protein and red fluorescent protein (RFP) were mixed in Agrobacterium buffer, and bacteria expressing GFP-CmFer1 and plasma membrane localized protein (PM) were mixed (OD600 = 0.8). The bacteria were injected into Nicotiana benthamiana plants. After 24 hours, tobacco leaves were cut, prepared into slides, and observed inverted under a laser confocal microscope Zeiss LSM710 (CarlZeiss, Oberkochen, Germany). GFP and RFP fluorescence signals were observed at excitation wavelengths of 488nm and 561nm, respectively. Co-localization was observed using the Merge function on the software. The results are shown in Figure 2. Figure 3 As shown, GFP-CmFer1 colocalized with PM on the plasma membrane of tobacco cells, while GFP colocalized with RFP in the nucleus and cytoplasm.

[0083] Example 5

[0084] Analysis of insect resistance of CmFer1

[0085] The tobacco leaves obtained in Example 2 were cut into discs (diameter = 3.5 cm), and then the discs transiently expressing CmFer1 and GFP genes were placed on the leftmost and rightmost sides of the bottom of a culture dish (diameter = 9 cm), and wet cotton wool was placed on the bottom of the leaves to keep them moist. Fifteen 2nd-instar fall armyworm larvae were selected from the middle of each leaf in the culture dish, and a total of 10 biological replicates were set up. The number of fall armyworm larvae on the leaves with different treatments was recorded after 12 hours and 24 hours, and the damage to the leaves was also recorded. The results are shown in Figure 2. Figure 4 As shown, the damage of tobacco leaves expressing GFP was more serious than that of SP7 leaves ( Figure 4 A in Figure 1). Compared to leaves transiently expressing SP7, Spodoptera frugiperda larvae prefer to feed on leaves transiently expressing GFP ( Figure 4 B) in.

[0086] Example 6

[0087] Evolutionary analysis and functional conservation analysis of CmFer1 protein

[0088] The homologous sequences of CmFer1 were searched in the Uniprot database and the amino acid sequences were sorted. The phylogenetic analysis of the Fer1 amino acid sequences of some Lepidoptera, Hemiptera, Coleoptera, Diptera, and Hymenoptera was performed using MEGA 11 software using the maximum likelihood (ML), neighbor-joining (NJ) and minimum evolution (ME) methods. The phylogenetic relationships were tested 1000 times using the bootstrap method. The results are as follows. Figure 5 As shown in A in Figure 3, the ferritins of insects from different orders clustered together and formed independent branches.

[0089] By searching the NCBI database, we obtained the coding sequences of the Chilo suppressalis ferritin CsFer1, Spodoptera frugiperda ferritin SfFer1, Nilaparvata lugens ferritin NIFer1, Spodoptera exigua ferritin SeFer1, and Helicoverpa armigera ferritin HaFer1. Specific primers were then designed based on these sequences for amplification. The primer sequences are as follows:

[0090] CsFer1-F (SEQ ID NO. 11): gacgagctgtacaagggtaccatgaagatcctgcttgc;

[0091] CsFer1-R (SEQ ID NO. 12): gcggactctagttcatctagattaaacatcgatacccat;

[0092] SfFer1-F (SEQ ID NO. 13): gacgagctgtacaagggtaccatgaaggccatcctcttt;

[0093] SfFer1-R (SEQ ID NO. 14): gcggactctagttcatctagattagatgtcgatgccaa;

[0094] NIFer1-F (SEQ ID NO. 15): gacgagctgtacaagggtaccatgtcgtcggttattac;

[0095] NIFer1-R (SEQ ID NO. 16): gcggactctagttcatctagattaagcttcattgctca;

[0096] SeFer1-F (SEQ ID NO. 17): gacgagctgtacaagggtaccatgaaggccatcctccttg;

[0097] SeFer1-R (SEQ ID NO. 18): gcggactctagttcatctagattagatgtcaatgccaa;

[0098] HaFer1-F (SEQ ID NO. 19): gacgagctgtacaagggtaccatgaaggccatcctccttg;

[0099] HaFer1-R (SEQ ID NO. 20): gcggactctagttcatctagattagatgtcgatgccaa.

[0100] According to the instructions of the homologous recombination kit, the above PCR product was gel-recovered and ligated into the pBIN-GFP vector after double enzyme digestion. After obtaining the recombinant overexpression vector, sequencing was performed. After sequencing was correct, the plasmid was transformed into GV3101 Agrobacterium competent cells. The positive Agrobacterium liquid was washed with Agrobacterium washing buffer and injected into tobacco (OD600 was adjusted to 0.4). The necrotic phenotype of the tobacco leaves was observed and photographed 3 days after injection. The results are as follows Figure 5 As shown in Figure B, except for the Chilo suppressalis ferritin CsFer1, which cannot induce cell necrosis, the other cloned insect ferritins can induce tobacco cell necrosis, indicating that the function of insect ferritins in inducing tobacco cell necrosis is conserved.

[0101] References

[0102] 1.ChenZD,LiuF,ZengMZ,WangL,LiuHM,SunYJ,Wang L,ZhangZC,ChenZY,XuYP,Zhang MM,Xia YQ,Ye WW,Dong SM,Govers F,Wang Y,Wang YC.Convergent evolution of immune receptors underpins distinct elicitinrecognition in closely relatedSolanaceous plants.Plant Cell.2023,35:1186–1201

[0103] 2.Cui JR,Bing XL,Tang YJ,Liu F,Ren L,Zhou JY,Liu HH,Wang MK,HoffmannAA,Hong XY.A conserved protein disulfide isomerase enhances plant resistanceagainst herbivores.PlantPhysiology.2023,191:660–678.

[0104] 3.Cui JR,Yao

[0105] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A rice leaf folder salivary protein CmFer1, characterized in that The rice leaf folder salivary protein CmFer1 includes any one of the following: 1) A protein comprising the amino acid sequence shown in SEQ ID NO. 1; 2) a protein obtained by replacing, deleting and / or adding one or more amino acid residues of the protein described in 1) and having an identity of more than 80% with the protein described in 1) and having the effect of improving plant insect resistance; 3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of the protein in 1) or the protein in 2) to a protein tag.

2. The homologous protein of the rice leaf folder salivary protein CmFer1 according to claim 1, characterized in that At least one of the proteins comprising the amino acid sequences shown in SEQ ID NOs. 2 to 5.

3. The gene CmFer1 encoding the rice leaf folder salivary protein CmFer1 according to claim 1.

4. The coding gene CmFer1 according to claim 2, characterized in that The coding gene CmFer1 includes any one of the following: (1) A gene comprising the nucleotide sequence shown in SEQ ID NO. 6; (2) A gene encoding the rice leaf roller salivary protein CmFer1 according to claim 1, obtained by replacing, deleting and / or adding one or more nucleotides of the gene described in (1); (3) A fusion gene obtained by connecting the 3' end and / or 5' end of the gene described in (1) or the gene described in (2) to a gene encoding a protein tag.

5. A recombinant vector, characterized in that The coding gene according to claim 3 or 4 is inserted.

6. A recombinant bacterium, characterized in that Comprising the recombinant vector according to claim 5.

7. Use of the rice leaf folder salivary protein CmFer1 according to claim 1, the homologous protein according to claim 2, the encoding gene CmFer1 according to claim 3 or 4, the recombinant vector according to claim 5, or the recombinant bacterium according to claim 6 in improving plant insect resistance or breeding insect-resistant plants.

8. The use according to claim 7, characterized in that The plants include tobacco and / or rice; the method of improving the insect resistance of plants includes improving the resistance of plants to pests; and the pests are fall armyworms.

9. A method for improving insect resistance of plants, characterized in that: The method comprises the following steps: overexpressing the rice leaf folder salivary protein CmFer1 according to claim 1, the homologous protein according to claim 2, or the encoding gene CmFer1 according to claim 3 or 4 in a plant.

10. The method for subcellular localization of the gene encoding CmFer1 in plants according to claim 3 or 4, characterized in that: The following steps are involved: The coding gene CmFer1 is transiently co-expressed with a plasma membrane localization marker gene to observe the subcellular localization of the CmFer1 protein.

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