A plant insect-resistant gene and its application and methods in plant insect resistance

Abstract

This invention discloses a plant insect-resistant gene and its application and method in plant insect resistance, belonging to the field of functional gene technology. The plant insect-resistant gene of this invention contains the conserved amino acid sequence shown in SEQ ID No. 1, or a conserved amino acid sequence with more than 80% homology to SEQ ID No. 1. This invention verified the function of NtCRF and AtCRF, both containing the conserved amino acid sequence of the GDSL motif, namely, that silencing or mutation significantly reduces resistance to insects; overexpression significantly increases plant resistance to insects. Therefore, it is speculated that genes containing the conserved amino acid sequence of the GDSL motif can be used to increase the difficulty for insects to feed on plant leaves, thereby improving plant insect resistance, and has broad application value.

Description

Technical Field

[0001] This invention belongs to the field of functional gene technology, specifically relating to a plant insect resistance gene and its application and method in plant insect resistance. Background Technology

[0002] Herbivorous insects first appeared on Earth approximately 350 million years ago. Since then, plants and insects have been in a constant cycle of attack and defense, evolving continuously. Based on this long-term relationship, plants have evolved a variety of strategies, including constitutive and induced defenses, to resist or evade insect attacks. On the one hand, plant structures, such as trichomes, cuticles, and cell walls, constitute natural physical barriers to defend against insect attacks; on the other hand, plants initiate a series of immune responses, such as calcium... 2+ Plants cope with insect feeding by accumulating endorphins and hormones. Correspondingly, insects have evolved different defense mechanisms and behaviors to adapt to plant evolution. Among them, piercing-sucking insects that feed on phloem, such as whiteflies and aphids, have become the most destructive pests, causing significant economic losses to major crops. They not only harm plants by sucking sap, causing them to wilt and die, but they can also transmit more than 200 plant viruses, leading to epidemics and outbreaks of crop viral diseases.

[0003] The feeding behavior of piercing-sucking insects is crucial to their population growth and reproduction. The mouthparts of piercing-sucking insects mainly consist of the labium, labium, mandibles, and maxillae. The labium elongates to form a proboscis, while the mandibles and maxillae are specialized to form stylets. The stylet is a vital organ for piercing-sucking insects to pierce plant tissue and extract nutrients from the phloem sieve tubes. After landing on a plant surface, piercing-sucking insects obtain phloem sap through a series of feeding behaviors, including surface exploration, stylet probing, and phloem feeding. In the process of reaching the phloem, the stylet must successively penetrate a series of tissues and organs, including the cuticle, epidermal cells, and mesophyll cells, to reach the phloem for successful feeding. The composition and structure of these physical barriers are essential for the insects' survival. However, the relationship between the probing behavior of piercing-sucking insect stylets and the composition and structure of these plant physical barriers remains unclear.

[0004] The host plant's defense response is active throughout the entire feeding phase of insects. To date, plant defenses against piercing-sucking insects have been extensively studied. Plants have evolved a variety of chemical and physical defense mechanisms to suppress insect feeding behavior. Plant hormone signal transduction and the production of plant secondary metabolites play a central role in plant responses to piercing-sucking insects. For example, salicylic acid (SA) and jasmonic acid (JA) exhibit both antagonistic and synergistic effects on resistance to piercing-sucking insects, which may depend on different plant genotypes. In addition, the plant cuticle, typically composed of cuticle and wax, is the first barrier for insect feeding. Reduction of the waxy cuticle in pea leaves is detrimental to the population growth of pea aphids. Pectin methyl esterase activity is a key factor affecting cell wall mechanical properties; inhibiting pectin methyl esterase activity can significantly alter aphid feeding preferences. Bph30, a resistance gene against the brown planthopper in rice, prevents the planthopper's stylet from reaching the phloem by strengthening the rice's sclerenchyma. Other mechanisms by which plants defend against insect infestations through physical defense require further investigation.

[0005] GDSL lipases (GDSL lipase proteins, GELPs) are widely distributed in all organisms and belong to a subfamily of lipases containing a conserved GDSL motif at the N-terminus of their proteins. This conserved GDSL motif contains four invariable important catalytic residues: serine (Ser), glycine (Gly), aspartic acid (Asn), and histidine (His). Therefore, GDSL lipases are also known as SGNH hydrolases. Currently, dozens to hundreds of GDSL lipase members have been identified in various plants, including 105 in Arabidopsis thaliana, 127 in Nicotiana tabacum, 80 in Solanum lycopersicum, 198 in Gossypium hirsutum, 240 in Brassica napus, 114 in Oryza sativa, and 113 in Pyrus bretschneideri. However, the biological functions of only a few GELPs have been studied in detail. Current research has found that the GDSL lipase gene EPITHIOSPECIFIERMODIFIER1 (ESM1) in Arabidopsis thaliana can enhance the plant's resistance to the white armyworm (Trichoplusia ni) by altering the hydrolysis of glucosinolates. Further research is needed to investigate the functions of GELPs in insect resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a plant insect-resistant gene and its application and method in plant insect resistance. The plant insect-resistant gene can improve the crop's own insect resistance and can control pest populations by interfering with the feeding behavior of insects.

[0007] The present invention provides a plant insect-resistant gene, which includes a conserved amino acid sequence as shown in SEQ ID No. 1, or contains a homologous conserved amino acid sequence having more than 80% homology with SEQ ID No. 1.

[0008] Preferably, when the plant insect-resistant gene is derived from tobacco, the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0009] Preferably, when the plant insect-resistant gene is derived from Arabidopsis thaliana, the amino acid sequence of the encoded protein is shown in SEQ ID No. 4.

[0010] This invention also provides the application of the above-mentioned plant insect-resistant genes in regulating plant resistance to diseases and pests.

[0011] Preferably, the regulation includes inhibiting the expression of the plant insect-resistant gene or knocking out the plant insect-resistant gene, which reduces the plant's resistance to diseases and pests; and inserting or overexpressing the plant insect-resistant gene, which increases the plant's resistance to diseases and pests.

[0012] Preferably, the pests include insects with piercing-sucking mouthparts.

[0013] Preferably, the insertion or overexpression is accomplished using genetic transformation; or the suppression is accomplished using virus-induced gene silencing.

[0014] The present invention also provides a method for improving plant resistance to diseases and pests, comprising inserting or overexpressing the above-mentioned plant insect-resistant genes into the genome of the target plant.

[0015] The present invention also provides the application of the above-mentioned plant insect-resistant genes or biomaterials related to the above-mentioned plant insect-resistant genes in the creation of plant germplasm with different insect-resistant phenotypes.

[0016] Beneficial Effects: This invention provides a plant insect-resistant gene (CRF) having a conserved amino acid sequence as shown in SEQ ID No. 1, or a conserved amino acid sequence with more than 80% homology to SEQ ID No. 1. In this embodiment, the plant insect-resistant gene derived from tobacco (Nicotiana tabacum) is named NtCRF, and the plant insect-resistant gene derived from Arabidopsis thaliana is named AtCRF. Both belong to the GDSL lipase family and both contain the conserved amino acid sequence of GDSLmotif.

[0017] In this invention, silencing the NtCRF gene using virus-induced gene silencing (VIGS) significantly reduced the resistance of tobacco plants to whiteflies and aphids. Furthermore, transgenic Arabidopsis thaliana and tomato overexpressing NtCRF were constructed using the Arabidopsis flower-dip transformation method and tomato tissue culture method. The transgenic Arabidopsis overexpressing NtCRF exhibited significant resistance to whiteflies and aphids, while the transgenic tomato overexpressing NtCRF also showed significant resistance to whiteflies. In addition, mutations in the NtCRF homolog AtCRF significantly reduced the resistance of Arabidopsis to whiteflies and aphids. Therefore, the CRF gene can significantly regulate the insect-resistant phenotype of plants.

[0018] In this embodiment of the invention, the electric penetration graph (EPG) technique was used to observe the feeding behavior of whiteflies on plant leaves. The results showed that the whitefly's probing time on transgenic Arabidopsis thaliana overexpressing NtCRF was significantly increased, indicating that it encountered increased resistance during feeding. That is, by interfering with the insect's feeding behavior, it achieves the purpose of controlling the pest population, providing a strategy that can be used to protect plants from piercing-sucking insects. In this way, the present invention can also use the CRF gene to genetically improve crops, thereby improving the crop's own insect resistance. Attached Figure Description

[0019] Figure 1 A comparison of NtCRF gene expression levels in control tobacco and tobacco with silenced NtCRF gene;

[0020] Figure 2 The results show the survival rate (left) and oviposition (right) of whiteflies on control tobacco and tobacco with silenced NtCRF;

[0021] Figure 3 A comparison of aphid numbers on control tobacco and tobacco with silenced NtCRF;

[0022] Figure 4 Figure 1 shows the results of PCR (left) and qRT-PCR (right) detection in positive Arabidopsis thaliana seedlings overexpressing NtCRF.

[0023] Figure 5 The results show the survival rate (left) and oviposition (right) of whiteflies on wild-type and NtCRF-overexpressing transgenic Arabidopsis thaliana;

[0024] Figure 6 A comparison of aphid numbers on wild-type and NtCRF-overexpressing transgenic Arabidopsis thaliana;

[0025] Figure 7 A comparison of the probing time (left) and feeding time (right) of whiteflies on wild-type and NtCRF-overexpressing transgenic Arabidopsis thaliana;

[0026] Figure 8 Figure 1 shows the results of Western blot (left) and qRT-PCR (right) detection of NtCRF-overexpressing transgenic tomato seedlings.

[0027] Figure 9 The results show the survival rate (left) and oviposition (right) of whiteflies on wild-type and NtCRF-overexpressing transgenic tomatoes;

[0028] Figure 10 Figure showing the amino acid sequence alignment results between NtCRF and Arabidopsis thaliana AtCRF;

[0029] Figure 11 The results show the survival rate (left) and oviposition (right) of whiteflies on wild-type and Atcrf mutant Arabidopsis thaliana;

[0030] Figure 12 A comparison of aphid numbers in wild-type and Atcrf mutant Arabidopsis thaliana;

[0031] Figure 13 The image shows the amino acid sequence alignment results of NtCRF with GDSL lipases from other species. Detailed Implementation

[0032] The present invention provides a plant insect-resistant gene, which contains a conserved amino acid sequence of FIFGDSLIDNGNNN as shown in SEQ ID No.1, or a conserved amino acid sequence having more than 80% homology with SEQ ID No.1.

[0033] In this invention, when the plant insect-resistant gene is derived from tobacco, it is named NtCRF, and the amino acid sequence of the encoded protein is preferably as shown in SEQ ID No. 2; the nucleotide sequence is preferably as shown in SEQ ID No. 3; when the plant insect-resistant gene is derived from Arabidopsis thaliana, it is named AtCRF, and the amino acid sequence of the encoded protein is preferably as shown in SEQ ID No. 4; the nucleotide sequence is preferably as shown in SEQ ID No. 5.

[0034] This invention also provides the application of the above-mentioned plant insect-resistant genes in regulating plant resistance to diseases and pests.

[0035] Although the embodiments of this invention only used the NtCRF and AtCRF genes for functional verification, such as knocking out or inhibiting the expression of the plant insect-resistant gene, which reduced the plant's resistance to diseases and pests, and inserting or overexpressing the plant insect-resistant gene, which increased the plant's resistance to diseases and pests, this should not be considered as the entire scope of protection of this invention. All homologous genes or family genes that have the conserved amino acid sequence of the NtCRF gene or similar amino acid sequences with more than 80% homology and produce similar effects should be included in the scope of protection of this invention.

[0036] The present invention also uses bioinformatics to predict that the NtCRF and AtCRF genes contain a conserved amino acid sequence containing GDSLmotif. This sequence is also relatively conserved in GDSL lipases of other species, suggesting that other GDSL family genes may also have certain insect-resistant effects.

[0037] The pests and diseases described in this invention preferably include insects with piercing-sucking mouthparts, and more preferably include whiteflies and aphids.

[0038] The present invention also provides a method for improving plant resistance to diseases and pests, comprising inserting or overexpressing the above-mentioned plant insect-resistant genes into the genome of the target plant.

[0039] The present invention does not specifically limit the method of insertion or overexpression, but preferably includes using genetic transformation to complete the insertion or overexpression. For example, in the embodiments, transgenic Arabidopsis thaliana overexpressing NtCRF is constructed by transforming Arabidopsis thaliana using the flower dipping method, and transgenic tomatoes expressing NtCRF are constructed using the tomato tissue culture method.

[0040] The present invention also provides a method for reducing plant resistance to pests and diseases, including inhibiting the expression of the above-mentioned plant insect resistance genes in the genome of the target plant, or knocking out the plant insect resistance genes.

[0041] The present invention does not specifically limit the method of knockout or inhibition, but preferably includes using a virus-induced gene silencing method to complete the inhibition, or mutating the plant insect-resistant gene.

[0042] The present invention also provides the application of the above-mentioned plant insect-resistant genes or biomaterials related to the above-mentioned plant insect-resistant genes in the creation of plant germplasm with different insect-resistant phenotypes.

[0043] The biomaterials related to plant insect-resistant genes described in this invention preferably include recombinant vectors.

[0044] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a plant insect-resistant gene and its application and method in plant insect resistance, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The effect of silencing NtCRF in common tobacco on fitness of whiteflies and aphids

[0047] 1. Obtaining common tobacco plants with silenced NtCRF gene

[0048] (1) The cDNA sequence of the NtCRF gene was obtained through... The plus One step PCR Cloning Kit homologous recombination kit was used to ligate the vector 2mDNA1, which was then transformed into E. coli by heat shock. After correct sequencing, the NtCRF-2mDNA1 plasmid was extracted using the AxyPrep Plasmid Miniprep Purification Kit, and then the plasmid was transformed into Agrobacterium by electroporation.

[0049] (2) When common tobacco plants reached the two true leaf stage, an NtCRF-2mDNA1 infectious clonal virus suspension was inoculated into the leaves using Agrobacterium-mediated transformation. After 18–20 days, total RNA was extracted from the inoculated leaves using the Trizol method. cDNA was synthesized via reverse transcription using the PrimeScript RT reagent Kit, and the silencing level of NtCRF in common tobacco was detected by quantitative real-time PCR. Primers were designed using Prime Primer 5.0 software and synthesized by Nanjing GenScript Biotech Co., Ltd. The results are as follows: Figure 1 As shown, after silencing NtCRF in ordinary tobacco using VIGS technology, the expression level of NtCRF decreased significantly compared with the control group.

[0050] NtCRF gene quantification primers:

[0051] qRTNtCRFF (SEQ ID No. 6): 5'-CCCTACTGGTCGTTTCTCCA-3';

[0052] qRTNtCRFR (SEQ ID No. 7): 5'-GCAGCAGCAGAAGCATAGTT-3'.

[0053] Internal reference gene quantification primers:

[0054] GAPDH F (SEQ ID No. 8): 5'-GCAGTGAACGACCCATTTATCTC-3';

[0055] GAPDH R (SEQ ID No. 9): 5'-AACCTTCTTGGCACCACCCT-3'.

[0056] 2. Effects of silencing the NtCRF gene in common tobacco on the fitness of whiteflies

[0057] Five female and five male whiteflies (within three days of initial emergence) were used as a replicate. Leaf traps were placed on the leaves of control tobacco and tobacco with silenced NtCRF. Ten whiteflies were then placed in the leaf traps. After 7 days, the survival rate and egg production of the whiteflies were recorded.

[0058] The results are as follows Figure 2 As shown, silencing NtCRF in ordinary tobacco using VIGS technology significantly improved the survival rate and egg production of whiteflies compared to the control group.

[0059] 3. The effect of silencing the NtCRF gene in common tobacco on aphid fitness.

[0060] Second-instar aphid nymphs were collected for bioassay. Three aphid nymphs were taken as a replicate. Leaf clamps were placed on tobacco leaves, and the three aphid nymphs were placed into the leaf clamps. The number of aphids was counted after 7 days.

[0061] The results are as follows Figure 3 As shown, silencing NtCRF in ordinary tobacco using VIGS technology significantly increased the number of aphids compared to the control group.

[0062] Example 2

[0063] Effects of NtCRF overexpression in Arabidopsis thaliana on fitness of whiteflies and aphids

[0064] 1. Construction of transgenic Arabidopsis thaliana overexpressing NtCRF

[0065] (1) The cDNA sequence of the NtCRF gene was obtained through... The pCambia1305-flag vector was ligated into the One Step PCR Cloning Kit and then transformed into E. coli by heat shock. After correct sequencing, the pCambia1305-NtCRF-flag plasmid was extracted using the AxyPrep Plasmid Miniprep Purification Kit and then transformed into Agrobacterium by electroporation.

[0066] 2) Take a small amount of Agrobacterium tumefaciens suspension (pCambia1305-NtCRF-flag) and incubate it in cannabidiol-resistant liquid LB medium at 28°C and 200 rpm on a shaker until the bacterial culture reaches OD. 600 The value is 1;

[0067] (3) Centrifuge at 25℃ and 6000rpm for 10min and collect the bacterial cells;

[0068] (4) Prepare a 5% sucrose solution for suspending the bacterial cells and adjust the OD. 600 The value is 0.8 to 1.0, then add 200 μL / L of silwet-77 and stir for 2 minutes;

[0069] (5) Immerse the Arabidopsis thaliana in the above solution for 20-30 seconds, culture in the dark at 22°C for 24 hours, then transfer it to the Arabidopsis thaliana culture room for culture, and immerse it again after one week (a total of 3 times), and then harvest the seeds.

[0070] (6) Positive seedlings were screened using hygromycin-resistant 1 / 2MS medium, and positive seedlings were detected by PCR and qRT-PCR.

[0071] PCR detection primers:

[0072] PCRNtCRF F (SEQ ID No. 10): 5'-ATGCAAGAAGTGGATGATGCA-3';

[0073] PCRNtCRF R (SEQ ID No. 11): 5'-AAGAGCAGCAAGCTGCTG-3'.

[0074] qRT-PCR detection primers:

[0075] qRTNtCRF F (SEQ ID No. 6): 5'-CCCTACTGGTCGTTTCTCCA-3';

[0076] qRTNtCRF R (SEQ ID No. 7): 5'-GCAGCAGCAGAAGCATAGTT-3'.

[0077] qRT-PCR internal reference gene quantification primers:

[0078] APX3 F (SEQ ID No. 12): 5'-CTCCGTTCTCTCATCGC-3';

[0079] APX3 R (SEQ ID No. 13): 5'-CAGAGATCGAGAGCGATC-3'.

[0080] The results are as follows Figure 4 As shown, four transgenic lines overexpressing NtCRF were obtained by Arabidopsis thaliana flower dipping method.

[0081] 2. Effects of NtCRF-overexpressing transgenic Arabidopsis on the fitness of whiteflies

[0082] Five female and five male whiteflies (within three days of initial emergence) were used as a replicate. Ten whiteflies were placed on Arabidopsis leaves, and the entire plant was covered with a Lock & Lock box (the Lock & Lock box has a 10cm diameter circular hole on the side and top, sealed with 120-mesh nylon netting for ventilation). After 7 days, the survival rate and egg production of the whiteflies were recorded.

[0083] The results are as follows Figure 5 As shown, the survival rate and egg production of whiteflies on NtCRF-overexpressing transgenic Arabidopsis thaliana lines 1, 2, and 5 were significantly lower than those of the control group.

[0084] 3. Effects of NtCRF-overexpressing transgenic Arabidopsis on aphid fitness

[0085] Wild-type and NtCRF-overexpressing transgenic Arabidopsis thaliana line5 were used for aphid bioassay. Three second-instar aphid nymphs were taken as a replicate. The three aphid nymphs were placed on Arabidopsis leaves, and the entire plant was covered with a Lock & Lock box (the Lock & Lock box has a 10cm diameter circular hole on the side and top, sealed with 120-mesh nylon mesh for ventilation). The number of aphids was counted after 7 days.

[0086] The results are as follows Figure 6 As shown, the number of aphids on NtCRF-overexpressing transgenic Arabidopsis thaliana was significantly lower than that in the control group.

[0087] 4. Effects of NtCRF-overexpressing transgenic Arabidopsis on feeding behavior of whiteflies

[0088] An 8-channel DC-EPG device (EPG Systems, Wageningen University, the Netherlands) was used to continuously record the feeding behavior of whiteflies on plant leaves, with each whitefly recorded for 8 hours. The whiteflies were connected to the DC-EPG device using a water-soluble silver conductive coating (Colloidal Silver, Wageningen University, the Netherlands). Simultaneously, the electrodes from the device were inserted into the soil to create a closed circuit between the whiteflies and the DC-EPG device. Stylet+ software was used to record and analyze the feeding behavior of the whiteflies.

[0089] The results are as follows Figure 7 As shown, the feeding time of whiteflies on NtCRF-overexpressing transgenic Arabidopsis thaliana did not change significantly compared with the control, but the probing time increased significantly compared with the control, indicating that the feeding resistance of whiteflies on NtCRF-overexpressing transgenic Arabidopsis thaliana is greater.

[0090] Example 3

[0091] Effect of NtCRF overexpression in tomatoes on the fitness of whiteflies

[0092] 1. Construction of transgenic tomatoes overexpressing NtCRF

[0093] (1) The cDNA sequence of the NtCRF gene was obtained through... The plus One step PCR Cloning Kit homologous recombination kit was used to ligate the vector pBWA(V)HS, which was then transformed into E. coli by heat shock. After correct sequencing, the pCambia1305-NtCRF-flag plasmid was extracted using the AxyPrep Plasmid Miniprep Purification Kit, and then the plasmid was transformed into Agrobacterium by electroporation.

[0094] (2) Wash tomato (Solanum lycopersicum cv Micro-Tom) seeds with sterile water for 2 min, disinfect with 75% alcohol for 1 min, soak in 5% hypochlorous acid solution for 5-8 min, wash with sterile water twice for 10 min each time, inoculate in sterile filter paper and air dry.

[0095] (3) Sow the disinfected seeds in the germination medium and culture them in the dark at 23°C for 2 days. After the seeds show white sprouts, place them in the dark at 23°C for 4-5 days under 16h / 8h light / dark conditions.

[0096] (4) After the tomato seedlings have fully expanded, use a scalpel to cut off the cotyledon petiole and cotyledon tip, leaving the middle part and cutting it into 2-3 segments. Inoculate the seedlings into the pre-culture medium and pre-culture at 23±2℃ for 1-2 days.

[0097] (5) Pick Agrobacterium and place it in the infection solution to prepare OD. 600 Agrobacterium resuspension at 0.2°C; infect for 10–15 min, then inoculate the dried explants into co-culture medium and incubate in the dark at 23±2°C for 2 days;

[0098] (6) Wash the co-cultured explants twice with 1g / L cephalosporin solution for 15min each time, then inoculate them into recovery medium and culture at 23℃ for 16h / 8h light / dark for 3 to 5 days.

[0099] (7) The recovered callus was inoculated into the selection medium and cultured at 23℃ for 16h / 8h light / dark for 15-30 days; the selected callus was inoculated into the differentiation medium and cultured at 23℃ for 16h / 8h light / dark for 30-40 days. When the seedlings to be differentiated grew to about 2-3cm, they were cut off from the callus and inoculated into the rooting medium and cultured at 23℃ for 16h / 8h light / dark for 10-15 days;

[0100] (8) Positive seedlings were detected by qRT-PCR and Western blot. Results are as follows: Figure 8 As shown, two transgenic lines overexpressing NtCRF were obtained by tomato tissue culture transformation.

[0101] qRT-PCR detection primers:

[0102] qRTNtCRF F (SEQ ID No. 6): 5'-CCCTACTGGTCGTTTCTCCA-3';

[0103] qRTNtCRF R (SEQ ID No. 7): 5'-GCAGCAGCAGAAGCATAGTT-3'.

[0104] qRT-PCR internal reference gene quantification primers:

[0105] Actin3 F (SEQ ID No. 14): 5'-GTCCTCTCCAGCCATCCA-3';

[0106] Actin3 R (SEQ ID No. 15): 5'-ACCACTGAGCACAATGTTACCG-3'.

[0107] 2. Effects of NtCRF-overexpressing transgenic tomatoes on the fitness of whiteflies

[0108] Five female and five male whiteflies (within three days of initial emergence) were used as a replicate. Leaf traps were placed on the leaves of control tomatoes and NtCRF-overexpressing transgenic tomatoes. Ten whiteflies were then placed in the leaf traps. After seven days, the survival rate and egg production of the whiteflies were recorded.

[0109] The results are as follows Figure 9 As shown, the survival rate and egg production of whiteflies on NtCRF-overexpressing transgenic tomatoes in lines 2 and 4 were significantly lower than those in the control group.

[0110] Example 4

[0111] The effect of the homolog of NtCRF, AtCRF, in Arabidopsis thaliana on fitness in whiteflies and aphids.

[0112] 1. Amino acid sequence comparison between NtCRF and Arabidopsis thaliana AtCRF genes

[0113] The amino acid sequences of NtCRF and Arabidopsis thaliana AtCRF were compared and analyzed using DNAMAN software.

[0114] The results are as follows Figure 10 As shown, NtCRF and AtCRF sequences have high sequence similarity, and the conserved amino acid sequence Block I also has extremely high similarity.

[0115] 2. Effects of Atcrf mutant Arabidopsis thaliana on the fitness of whiteflies

[0116] (1) Purchase the Atcrf Arabidopsis mutant (SALK_022761C) from the Arashare website (https: / / www.arashare.cn);

[0117] (2) Take 5 female whiteflies and 5 male whiteflies (within 3 days of initial emergence) as a replicate. Place 10 whiteflies on the leaves of wild-type and Atcrf mutant Arabidopsis thaliana, and cover the entire plant with a Lock & Lock box (the Lock & Lock box has a circular hole with a diameter of 10cm on the side and top, which is sealed with 120-mesh nylon netting for ventilation). After 7 days, the survival rate and egg production of the whiteflies are counted.

[0118] The results are as follows Figure 11 As shown, the survival rate and egg production of whiteflies on Atcrf mutant Arabidopsis leaves were significantly higher than those of the control group.

[0119] 3. Effects of the Atcrf mutant Arabidopsis thaliana on aphid fitness

[0120] Three aphid nymphs (second instar) were taken as a replicate. The three aphid nymphs were placed on the leaves of wild-type and Atcrf mutant Arabidopsis thaliana. The entire plant was covered with a Lock & Lock box (the Lock & Lock box has a circular hole with a diameter of 10cm on the side and top, which is sealed with 120-mesh nylon netting for ventilation). The number of aphids was counted after 7 days.

[0121] The results are as follows Figure 12 As shown, the number of aphids on the leaves of the Atcrf mutant Arabidopsis thaliana was significantly higher than that of the control group.

[0122] 4. Comparison of amino acid sequences of NtCRF with GDSL lipases from other species

[0123] The amino acid sequences of NtCRF were compared and analyzed with those of GDSL lipases from other species, such as BnGLIP1 (Brassica napus), CaGL1 (Capsicumannuum), SlGDSL1 (Solanum lycopersicum), and AtCRF (Arabidopsisthaliana), using DNAMAN software.

[0124] The results are as follows Figure 13 As shown, the amino acid sequence of GDSL lipase Block I is also relatively conserved in other species, indicating that GDSL lipases in other species may also be involved in insect resistance.

[0125] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of tobacco insect-resistant genes in regulating tobacco resistance to diseases and pests, characterized in that, The regulation involves inserting or overexpressing the tobacco insect-resistant gene in tobacco, which enhances the tobacco's resistance to pests and diseases; the pests and diseases are piercing-sucking insects. The amino acid sequence of the protein encoded by the tobacco insect-resistant gene is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, This includes using genetic transformation methods to complete the insertion or overexpression.

3. A method for improving tobacco's resistance to diseases and pests, characterized in that, This includes inserting or overexpressing tobacco insect-resistant genes into the tobacco genome; The amino acid sequence of the protein encoded by the tobacco insect-resistant gene is shown in SEQ ID No. 2; the insect is a piercing-sucking insect.

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