CN115896118B - Brown planthopper salivary protein NlG gene and protein for inducing plant to generate resistance and application thereof - Google Patents
Brown planthopper salivary protein NlG gene and protein for inducing plant to generate resistance and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention discloses a brown planthopper salivary protein NlG gene and protein for inducing plants to generate resistance and application thereof, wherein the brown planthopper salivary protein gene is NlG, and the nucleotide sequence of the brown planthopper salivary protein gene is shown as SEQ ID NO. 1. The present invention identifies a salivary protein gene NlG from brown planthopper (Nilaparvata lugens) that encodes a protein that triggers HR, ROS, and callose deposition, thereby inducing plant resistance, including insect resistance and disease resistance. The brown planthopper salivary protein NlG has the value developed into a plant immunity inducer, provides theoretical support for preventing and controlling plant diseases and insect pests, can be directly introduced into crops to prepare transgenic plants, and provides a novel transgenic disease-resistant strategy.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to brown planthopper salivary protein NlG gene and protein for inducing plants to generate resistance and application thereof.
Background
During the long-term co-evolution of plants and insects, insects have evolved a number of weapons, such as effector proteins (effectors), that attack the host in order to better feed on the plant. Insects secrete these effector proteins into the plant body in order that they better feed on the host plant, however, with co-evolution of the plant, some effector proteins are recognized by regulatory disease-resistance-related genes that the plant itself evolved. Recognition of effector proteins typically triggers a local resistance response, termed Hypersensitivity (HR), characterized by rapid cell death at the site of infection. This apparent HR response is also an important signal as a trigger for plant immunity. Reactive oxygen species accumulation is also a key signal triggering plant immune responses and can also be used as a signal marker for triggering plant defensive responses. Inoculation of pathogens or insects using agrobacterium-mediated transient expression systems is the most straightforward method to assess whether genes are capable of inducing plant resistance.
ROS accumulation plays a very important role in plant defense reactions, and it is involved in regulating stomatal movement, promoting apoptosis, and acting as a second messenger to activate and regulate the expression of various defense-related genes in plants at the transcriptional level. The deposition of plant callose can strengthen plant cell walls to prevent invasion of pathogenic microorganisms, and can also block a screen pipe to prevent sucking insects from eating phloem juice, so that the purposes of disease resistance or insect resistance are achieved. Plant immunity elicitors can be classified into biological and non-biological sources, and biological sources of immunity elicitors are mainly identified and purified from bacteria, fungi and viruses. Currently, insect-derived immune-induced antigen proteins are still relatively few.
Disclosure of Invention
The invention aims to: aiming at the defect of insufficient crop insect-resistant gene resources in the prior art, the invention provides the brown planthopper saliva protein gene for inducing plants to generate resistance, the saliva protein NlG14 from brown planthoppers (Nilaparvata lugens) is obtained through screening, and the protein promotes the resistance to pests and pathogens by triggering plant allergic reaction (HR), callose deposition and Reactive Oxygen Species (ROS) accumulation, so that crop production is protected, and the brown planthopper saliva protein gene NlG capable of inducing plants to generate resistance is proposed for the first time.
The invention also provides brown planthopper salivary proteins NlG for inducing plants to generate resistance, an expression vector and application.
The technical scheme is as follows: in order to achieve the aim, the invention provides a brown planthopper saliva protein gene for inducing plants to generate resistance, wherein the brown planthopper saliva protein gene is NlG, and the nucleotide sequence of the brown planthopper saliva protein gene is shown as SEQ ID NO. 1.
The gene-encoded brown planthopper salivary protein NlG14 is characterized in that the amino acid sequence of the brown planthopper salivary protein NlG14 is shown as SEQ ID NO. 2.
The invention relates to an expression cassette, a recombinant expression vector, a transgenic cell line or a transgenic recombinant strain of a brown planthopper salivary protein gene.
Wherein the starting vector of the recombinant expression vector is an expression vector PBINGFP2.
Preferably, a recombinant expression vector pBINGFP2: nlG14 of the NlG gene is constructed. The method is characterized in that a plant expression vector PBINGFP2 is taken as an initial vector, and NlG gene is inserted into KpnI and BamHI cleavage sites to obtain the vector.
The NlG gene or the protein or the expression cassette, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacterium are applied to inducing plants to generate resistance.
Wherein the resistance includes insect resistance and disease resistance.
Preferably, the use of inducing local necrosis and reactive oxygen species accumulation in plants to induce resistance in plants is provided.
The gene or the protein or the expression cassette, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacterium are applied to the preparation of the plant immunity elicitor.
The invention also provides a plant immunity elicitor, which contains the fermentation broth of the brown planthopper salivary protein NlG recombinant bacteria.
The gene or the protein or the expression cassette, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacteria are applied to cultivation of disease and insect resistant crop varieties.
Wherein, the genes, proteins, expression cassettes, recombinant expression vectors, transgenic cell lines or transgenic recombinant bacteria are introduced into plants, and positive transformed plants are obtained through resistance screening, thus obtaining disease and insect resistant crop varieties.
Further, the gene, protein, expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacteria are introduced into plants to trigger HR and ROS to induce the plants to generate resistance.
The saliva protein gene derived from brown planthoppers (Nilaparvata lugens) can trigger plant allergic reaction (HR), callose deposition and Reactive Oxygen Species (ROS) accumulation through screening, and can induce plants to generate resistance through objective evaluation of an agrobacterium-mediated transient expression system. The method has the advantages of activating HR, promoting ROS accumulation, promoting callose deposition, triggering plant immunity, inducing plant to generate disease resistance reaction, and being beneficial to research and development of plant immunity inducer. The gene has powerful function, and may be used in developing plant immunity inducing and resisting agent, and the gene may be introduced directly into plant with transgenic technology to develop and culture disease and pest resisting variety. The development of crop disease and insect resistant varieties is the most economical and effective method for pest control, and can reduce pesticide use and reduce environmental pollution risk while efficiently controlling pest. The brown planthopper salivary protein NlG is found to have the characteristic of inducing plants to generate resistance for the first time, and a molecular biological mechanism for inducing the plants to generate the resistance is defined, so that the development of the genes and the proteins with the immunity resistance and the creation of transgenic plants are facilitated.
The invention takes model plant Nicotiana benthamiana as a test object, the Nicotiana benthamiana can be used as a plant representative, and the mechanism of regulating and controlling plant defense response of the brown planthopper potential salivary protein NlG is researched through an agrobacterium-mediated transient expression system. The results of fig. 1 indicate that transient expression of NlG14 can cause plant allergic reactions (HR) as well as Reactive Oxygen Species (ROS) accumulation. The invention also detects the influence of transient expression of NlG on the plant callose deposition by an aniline blue staining method. The results in fig. 2 demonstrate that transient expression of NlG14 can significantly enhance callose deposition in plants. The present invention also evaluates plant resistance to pest by inoculating pathogenic bacteria or insects onto the leaf of the benthamiana of transient expression NlG14. The results of fig. 3 show that the transient expression of NlG14 effectively inhibits phytophthora capsici infection and spodoptera frugiperda feeding.
The brown planthopper salivary protein NlG has the function of inducing plants to generate resistance for the first time, and the molecular biological mechanism of inducing plants to generate resistance is clear, so that the development of the genes and the proteins with resistance in immunity and the creation of transgenic plants are facilitated.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the invention identifies a salivary protein gene NlG from brown planthoppers (Nilaparvata lugens), and firstly proposes the application of the brown planthoppers salivary protein gene NlG and protein NlG14 in inducing plants to generate insect resistance and disease resistance, inserting the gene NlG14 into a plant expression vector PBINGFP2, and introducing the vector into plants, so that obvious HR reaction, ROS accumulation and callose deposition can be generated, and plant immunity is stimulated.
The present invention expresses NlG Benshiyan, has excellent feeding inhibiting and infection inhibiting effect on various pests and pathogenic bacteria, and the NlG protein gene has value developed into plant immunity inducing and resisting agent and provides theoretical support for preventing and controlling plant diseases and pests. Meanwhile, the plant can be directly introduced into crops to prepare transgenic plants, and a novel transgenic disease-resistant strategy is provided.
Drawings
FIG. 1 shows NlG protein-triggered plant HR and ROS; wherein A is that the transient expression of agrobacterium and the injection of the benthamiana leaf pieces induce an HR phenotype; b is that the agrobacterium transient expression injection of the benthamiana tobacco leaves can induce active oxygen accumulation;
FIG. 2 shows NlG protein-induced plant leaf callose accumulation; wherein the numbers represent the average number of callose spots from three leaf discs;
FIG. 3 is NlG for inducing plant resistance; wherein A is that the agrobacterium transient expression NlG gene can improve the resistance of plants to phytophthora capsici on tobacco; b is the relative area statistics of the disease spots of the agrobacterium induced disease resistance phenotype; c is the transient expression NlG gene of agrobacterium in tobacco to raise the resistance of plant to spodoptera frugiperda; d is the larval count of the Agrobacterium-induced insect-resistant phenotype.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The raw materials, reagents and the like used in the examples of the present invention are commercially available, or the same type of raw materials are commercially available.
Example 1
1. Experimental insect
The test insect brown planthopper (Nilaparvata lugens) is provided by a pesticide pharmacology and neurophysiology laboratory at Nanjing university, and is subjected to indoor (temperature: 27+/-1 ℃ and humidity: 70% +/-10% and illumination: L: D=16 h:8 h) passage and breeding on Xiushui11 rice.
2. Preparation of test Benshi tobacco seedlings
Nicotiana benthamiana (Nicotiana benthamiana) (Hua Vietnam Biolimited, beijing) was sown in disposable plastic cups (200 mL) containing vermiculite and grown in a 16h light/8 h dark greenhouse. Growing the plant for 7 days, and transplanting the plant to vermiculite black soil with the volume ratio of 5 after growing two true leaves: 1 were grown in the culture soil for 30 days, leaves at the same 3,4,5 leaf positions were taken as the expression test genes, and then observation was continued for 7 days as to whether the leaf phenotype had obvious HR.
3. Extracting the RNA of brown planthopper (Nilaparvata lugens) and obtaining the cDNA of the target gene.
30 head five-year brown planthoppers (Nilaparvata lugens) were collected and Trizol was used according to Invitrogen corporation's instructions TM reagent extracts total RNA. Genomic DNA was removed using PrimeScript RT Reagent Kit according to TaKaRa, followed by reverse transcription to synthesize cDNA. PCR amplification was performed using the cDNA as a template and the corresponding upstream and downstream primers (primer sequences are shown in Table 1)
TABLE 1 primer sequences
| name | sequence |
| NlG14-F | ATGCTACTGAGGAAGATTGTTT |
| NlG14-R | CTACAGCTCATCTTTCTCGTTATT |
The PCR reaction system is as follows: 12.5. Mu.L 2X Phanta Master Mix; 2. Mu.L (10 μm) of primer; 1 μl of cDNA template; sterile water was made up to 25 μl.
The reaction procedure is: pre-denaturation at 95℃for 5 min; denaturation at 95℃for 15s, annealing at 55℃for 15s, elongation at 72℃for 60s,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
The PCR amplified product is subjected to agarose gel electrophoresis, recovery and sequencing to confirm that the obtained sequence is the nucleotide sequence of the NlG gene. The nucleotide sequence of the NlG gene is shown as SEQ ID NO. 1. The amino acid sequence of the protein coded by the NlG gene is shown as SEQ ID NO. 2. Subsequent studies have found that it has a powerful function of inducing necrosis and active oxygen accumulation in plants.
Example 2
Construction of pBINGFP2: nlG14 expression vector
The full-length sequence containing no signal peptide was amplified by PCR using the cDNA of example 1 as a template and using the upstream and downstream primers added with the homology arms of the plasmid (primer sequences are shown in Table 2). The PCR amplified product was subjected to agarose gel electrophoresis and recovered (the method was the same as in step (3) of example 1), to obtain the target gene.
TABLE 2 primer sequences
The pBINGFP2 (supplied from the Phytophthora interaction laboratory at Nanjing university of agriculture, tianqiao S, zhenchuan M, danyu S, et al, an Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters [ J ]. Plos Pathogenens, 2015,11 (12): e 1005348) empty vector plasmid was digested with KpnI and BamHI; the fragment of interest was ligated to the vector using the homologous recombinase Exnase II (cat# C112) from Norpraise.
The reaction system is as follows: 4 μL of 5 xCE II Buffer;2 μL of ExnaseII; 1. Mu.L of the above PCR recovered product; 4. Mu.L of the digested empty vector; sterile water was made up to 10 μl.
The reaction procedure is: the reaction was carried out at 37℃for 30min. The ligation product is transformed into E.coli DH5 alpha, transformants are screened on Kan resistant medium, and positive clone extraction plasmids are picked up for colony PCR identification. Sequencing the identified positive clone, and obtaining the PBINGFP2: nlG14 expression vector correctly, wherein the sequence of the PBINGFP2: nlG expression vector is shown as SEQ ID NO. 3.
Example 3
And (5) transforming agrobacterium with the expression vector and performing functional verification.
Agrobacterium transformation with PBINGFP2:NlG14 expression vector
The expression vector PBINGFP2: nlG14 was transformed into Agrobacterium cells GV3101 (available from the bang corporation) by electrotransformation. The specific process is as follows: the expression vector constructed in example 2 was added to agrobacterium competent, gently mixed and transferred to a sterile cuvette for shock transformation. After completion of the electric shock, the Agrobacterium competent cells were added to 700. Mu.L of LB liquid medium at a volume ratio of 10%, and cultured with shaking at 28℃for 2 hours at 200 rpm. mu.L of the bacterial liquid was aspirated, and the resulting mixture was spread evenly on a solid LB medium plate containing 50mM kanamycin and 25mM rifampicin, and cultured upside down at 28℃for 48 hours. And (3) picking a single colony for colony PCR to obtain positive clones, namely the agrobacterium containing the expression vector of PBINGFP2: nlG14.
2. Transient expression of PBINGFP2: nlG14, PBINGFP2 and PBI121:INF1 in Nicotiana benthamiana
Agrobacterium containing PBINGFP2: nlG14 obtained in step (1), agrobacterium containing empty PBINGFP2 (method same as step (1)) and Agrobacterium containing the PBI121:INF1 vector (wherein the PBI121 vector is a conventional plant expression vector, and the PBI121 expression INF1 protein is provided by the plant-phytophthora interaction laboratory at the university of Nanjing agriculture, dong Y, jing M, shen D, et al, the mirid bug Apolygus lucorum deploys a glutathione peroxidase as a candidate effector to enhance plant susceptibility [ J ]. Journal of Experimental Botany,2020 (9): 9) were inoculated into liquid LB medium containing 50mM kanamycin and 25mM rifampicin, respectively, and shake cultured at 28℃at 230rpm for 20 hours. The bacterial solution was collected, centrifuged at 5000rpm for 3min and washed once with permeation buffer (10mM MgCl2,500mM MES,100mM acetosyringone). Finally, the bacterial solution is diluted by osmotic buffer until the OD600 value is 0.4. The leaf of Nicotiana benthamiana containing different genes is respectively injected with a 1mL injector (the area of each leaf is guaranteed to be the same after injection and infiltration, and the leaf is generally saturated), each at least 6 repeats are performed, the experiment is repeated three times, and the result is shown in figure 1.
3. Reactive Oxygen Species (ROS) accumulation assay
After 48h of injection of all the treated genes, the cells were soaked in DAB dye solution (1 mg/mL DAB,10mM Na2HPO4) at 25℃for 6h in the absence of light, then decolorized with 95% ethanol, finally the phenotype was recorded by photographing with a camera, and the experimental results are shown in FIG. 1. The results of FIG. 1 show that transient expression of gene NlG14 can cause plant allergic reactions (HR) and Reactive Oxygen Species (ROS) accumulation, and that positive control INF1 also shows HR reactions and ROS accumulation, but negative control GFP does not change at all.
4. Callose deposition assay
After 48 injections, the leaf discs were decolorized with 95% ethanol and the bleached samples were subjected to ddH 2 O-washing with dye solution (70 mM KH) 2 PO 4 And 0.05% aniline blue, pH 9) for 1h at room temperature, the stained leaves were attached to an anti-fluorescence quenching capper, photographed by observation with a fluorescence inversion microscope, the number of callose precipitates was counted with imageJ software, the experiment was repeated three times, and the experimental results are shown in FIG. 2. The results of fig. 2 show that transient expression of gene NlG14 can significantly enhance callose deposition in plants, while demonstrating expression of NlG protein.
5. Detection of disease resistance of Benshi smoke by transient expression of PBINGFP2: nlG14 protein
The leaf of Benshi tobacco which expresses NlG14 protein is cut off and placed in a tray which is moist with filter paper. Punching holes on the growth edges of the phytophthora capsici tablet by using a puncher, and arranging the punched fungus discs on the left and right sides of the leaf tobacco sheet in a bilateral symmetry way with the fungus faces facing downwards. The trays were placed in a 25℃incubator, dark, for 36h. The leaves were removed and then irradiated with a hand-held uv fluorometer. The darkened area is the infected area of phytophthora capsici. Leaf surfaces expressing GFP proteins are used as controls, the relative infection area is measured by Image J software, and the effect of the two proteins on resistance to Benshi smoke is compared. The NlG protein from brown planthoppers was found to significantly improve the resistance of Nicotiana benthamiana to Phytophthora capsici (as shown in FIG. 3), while the same method as described above was used to test the resistance of other pathogenic bacteria, including Phytophthora parasitica, sclerotinia sclerotiorum, ralstonia solanacearum, pseudomonas syringae, alternaria alternata, pectobacterium carotovorum, etc. (as shown in Table 1), demonstrating that NlG protein significantly improves the resistance of Nicotiana benthamiana to the pathogenic bacteria.
TABLE 1 inhibition of pathogenic bacterial infection by transiently expressed PBINGFP2: nlG14 protein
6. Detection of resistance to Benshi tobacco insects by transient expression of PBINGFP2: nlG14 protein
According to the method in the step (2), GFP or GFP: nlG is transiently expressed in leaves of Nicotiana benthamiana by using a transient expression system in Nicotiana benthamiana, and the leaves are cut off after 24 hours and placed in a tray. The stems were moisturized with moist cotton, 8 spodoptera frugiperda larvae of 2 ages were placed in the middle of the leaves, and after 12h and 24h, two leaves were counted for 16 replicates, respectively. The expression of NlG protein was found to significantly inhibit feeding of spodoptera frugiperda (as shown in fig. 3), while resistance to other pests including cotton bollworms, prodenia litura, asparagus caterpillar, corn borers, meadow moth, peach aphids, cotton aphids, bemisia tabaci, etc. (as shown in table 2) was tested using the same method described above, indicating that the expression of NlG protein significantly inhibited feeding of the above pests.
TABLE 2 inhibition of pest hazard by transient expression of PBINGFP2: nlG14 protein
Claims (9)
1. A first partThe brown planthopper saliva protein gene for inducing plant to generate resistance is characterized in that the brown planthopper saliva protein gene isNlG14The nucleotide sequence is shown as SEQ ID NO. 1.
2. A brown planthopper salivary protein NlG14 encoded by the gene of claim 1, wherein the amino acid sequence of the brown planthopper salivary protein NlG is shown in SEQ ID No. 2.
3. An expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacterium comprising the brown planthopper salivary protein gene of claim 1.
4. A recombinant expression vector according to claim 3, wherein the starting vector of the recombinant expression vector is the expression vector pbinfp 2.
5. Use of a NlG gene as claimed in claim 1 or a protein as claimed in claim 2 or an expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacterium as claimed in claim 3 for inducing resistance in plants, including insect resistance and disease resistance.
6. The use according to claim 5, characterized by the use of inducing local necrosis and reactive oxygen species accumulation in plants for inducing resistance in plants.
7. Use of a gene according to claim 1 or a protein according to claim 2 or an expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant bacterium according to claim 3 for the preparation of a plant immunity-inducing agent.
8. Use of a gene according to claim 1 or a protein according to claim 2 or an expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant strain according to claim 3 for the cultivation of disease and pest resistant crop varieties.
9. The use according to claim 8, wherein said gene, protein, expression cassette, recombinant expression vector, transgenic cell line or transgenic recombinant strain is introduced into a plant, and a positive transformed plant is obtained by resistance selection to obtain a disease and pest resistant crop variety.
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| CN113337503A (en) * | 2021-04-09 | 2021-09-03 | 广东省农业科学院植物保护研究所 | Application of brown planthopper NLSP7 as target spot in prevention and treatment of brown planthopper |
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| CN113337503A (en) * | 2021-04-09 | 2021-09-03 | 广东省农业科学院植物保护研究所 | Application of brown planthopper NLSP7 as target spot in prevention and treatment of brown planthopper |
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| A Salivary Odorant-Binding Protein Mediates Nilaparvata lugens Feeding and Host Plant Phytohormone Suppression;Hao Liu等;INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES;第22卷(第9期);第1-15页 * |
| 褐飞虱效应子PIB14的研究;郭琴;中国优秀硕士学位论文全文数据库;D046-83 * |
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