Plasmid localization guide peptide recA1, primer pair, method for delivering nematicidal protein transgene and application thereof

Abstract

The present application discloses a plastid-localized guide peptide recA1, a primer pair, a method for delivering a nematicidal protein transgene, and its application, and relates to the field of plant protection technology. The guide peptide recA1 has a nucleotide sequence as shown in SEQ ID NO.2. The primer pair used for amplification to prepare the plastid-localized guide peptide recA1 includes a forward primer RecA1‑F1 and a reverse primer RecA1‑R1. The present application innovatively uses plastids to deliver the nematicidal protein Cry5Ba2, which effectively improves the resistance of plants to root-knot nematodes, inhibits the root knot formation of root-knot nematodes and the number of offspring eggs, indicating that the plastid-delivered anti-nematode protein technology can effectively improve the resistance of plants to root-knot nematodes, and the plastid-delivered anti-nematode protein technology has laid a solid foundation for the prevention and control of root-knot nematodes, and provides an effective way to prevent and control root-knot nematodes.

Description

Technical Field

[0001] The present application relates to the technical field of plant protection, and in particular to a plastid-localized guide peptide recA1, a primer pair, a method for delivering a nematicidal protein transgene, and applications thereof. Background Art

[0002] Plant-parasitic nematodes (PPNs) are one of the important pathogens of plant infectious diseases. The damage they cause to plants exceeds that of bacteria and viruses, and is second only to fungal diseases. More than 70% of the huge losses caused by plant-parasitic nematodes to crops can be attributed to root-knot nematodes. Root-knot nematode disease is a global disease. From a global distribution perspective, the disease is mainly distributed in temperate, subtropical and tropical regions, and occurs in almost all countries and regions.

[0003] Common root-knot nematodes include southern root-knot nematode (M. incognita), Java root-knot nematode (M. javanica) and peanut root-knot nematode (M. arenaria). Among them, southern root-knot nematode is the dominant population, with a wide distribution and serious damage. The above three root-knot nematodes all belong to the class Nematoda and the genus Meloidogyne. It is one of the main diseases of the underground roots of plants. It not only affects the growth and development of plants and reduces their quality. It also aggravates the occurrence of soil-borne pathogens. In recent years, with the continuous expansion of the scale of urban landscaping construction, a large number of new plant varieties have been introduced. According to the investigation of road green belts, street gardens, parks, greenhouses, and nursery bases, root-knot nematode disease has a tendency to increase.

[0004] The main damage of root-knot nematodes to plants is in the roots. When the root-knot nematode disease is serious, the symptoms of the above-ground parts of the plant are easily confused with other diseases, so people often ignore the damage of root-knot nematodes to plants. Root-knot nematodes are only infectious during the second-instar larvae (J2). J2 pierces the root tissue of the plant through the mouth needle and migrates along the cortex toward the root tip. After reaching the elongation zone, it turns around and migrates toward the upper end along the vascular bundle, so that the root-knot nematodes avoid the highly lignified Casparian strip.

[0005] Root-knot nematodes pierce the cell wall of plants through their mouth needles, inject secretions from their esophageal glands into plant cells and absorb nutrients from plants. In this parasitic process, proteins secreted by different organs of nematodes (lateral organs, esophageal glands, and epidermis) play important roles, such as escaping the host's defense response, assisting the movement of nematodes in root tissues, and inducing the formation and maintenance of feeding sites.

[0006] As the secretions are injected into the cells, the root-knot nematodes establish feeding sites in the plant roots, interfering with the normal growth and development of the cells. The plant cells undergo multiple nuclear divisions without cytoplasm division, and the multinucleated giant cells that are eventually formed are more than 100 times larger than normal cells. The highly differentiated giant cells have dense cytoplasm and vigorous metabolism, and are the only source of nutrition for the root-knot nematodes throughout their life cycle. After the feeding sites are established, the nematodes continue to develop and eventually develop into mature pear-shaped females. The females, together with the giant cells and secondary conducting tissues, form huge root knots, which interrupt the arrangement of normal vascular tissues, force the vessels to connect in a circuitous way or form secondary conducting structures in the cortex, causing physical damage to the internal tissues of the roots, and hindering and interfering with the normal transportation of water, mineral elements and organic matter.

[0007] According to the endosymbiotic theory of eukaryotic evolution, plant plastids evolved from cyanobacteria, but in the long process of evolution, most of their genes have been transferred to the nuclear genome. Chloroplasts are mainly found in plant leaves, green stems and mature fruits. They belong to the same plastids as chromoplasts (mature fruits, flowers), amyloplasts (starch storage organs), leucoplasts (flowers, roots, non-green stems), and oleoplasts (fat and oil storage organs). They are variants in different organs and different stages of development. They have different functions and morphologies, but they all develop from proplastids and have the same genetic material. In particular, the leucoplasts of plants are important energy storage organelles in plant roots.

[0008] There have been many reports and applications of transgenic crops expressing nematicidal proteins from Bacillus thuringiensis to provide crops with resistance to pests. However, there is currently no plastid-delivered anti-nematode protein technology for the control of root-knot nematodes. Summary of the invention

[0009] In response to the problems existing in the prior art, the inventors of this application creatively disclosed a plastid-localized guide peptide recA1, a primer pair, a method for delivering a nematicidal protein transgene and its application, which provides a new and effective method for plant protection and the control of root-knot nematodes by using the guide peptide to locate the nematicidal Bt protein in the plastid in plants to resist root-knot nematodes.

[0010] To this end, the embodiments of the present application at least disclose the following technical solutions:

[0011] In a first aspect, the present application discloses a plastid localization leader peptide recA1, wherein the leader peptide recA1 has a nucleotide sequence as shown in SEQ ID NO.2.

[0012] In the second aspect, the embodiments of the present application disclose a primer pair for amplifying and preparing the plastid localization leader peptide recA1 as described in the first aspect, the primer pair comprising a forward primer and a reverse primer, the forward primer RecA1-F1 is 5'-cgccccgcggaaagcttaattccaatccca-3'; the reverse primer RecA1-R1 is 5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3'.

[0013] In a third aspect, an embodiment of the present application discloses a method for delivering a nematicidal protein transgene, the method comprising recombinantly expressing a key gene for inhibiting root-knot nematodes in a plant by Agrobacterium-mediated nuclear transformation technology combined with a plastid-localized guide peptide recA1.

[0014] In a fourth aspect, the embodiments of the present application further disclose a use of a transgenic plant delivering a nematicidal protein obtained by the method described in the third aspect in the preparation of a plant resistant to root-knot nematodes.

[0015] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0016] This application innovatively uses plastid delivery (localizing the plant-expressed anti-nematode protein in the plastid through the plastid localization guide peptide recA1) of the nematode-killing protein Cry5Ba2, which effectively improves the plant's resistance to root-knot nematodes, inhibits the root knot formation of root-knot nematodes and the number of eggs in the offspring, indicating that the technology of plastid delivery of the anti-nematode protein Cry5Ba2 can effectively improve the plant's resistance to root-knot nematodes.

[0017] The technology of plastid delivery of anti-nematode protein Cry5Ba2 in the present application lays a solid foundation for the prevention and control of root-knot nematodes and provides an effective way for the prevention and control of root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Western blot image of plant root-knot nematode feeding on plastid protein provided in the examples of the present application;

[0019] Figure 2 is a vector map of the plant expression vector provided in the examples of this application;

[0020] Figure 3 This is a western blot image comparing the Cry5Ba2 protein levels in the plant expression vectors WY1 and WY24 provided in the examples of the present application;

[0021] Figure 4 This is a diagram of the root knot number of root-knot nematodes according to the results of the examples of the present application;

[0022] Figure 5 This is a graph showing the number of root-knot nematode eggs according to the results of the examples of the present application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0026] The present application discloses a plastid localization leader peptide recA1, wherein the leader peptide recA1 has a nucleotide sequence as shown in SEQ ID NO.2.

[0027] The present application also discloses a primer pair for amplifying and preparing the plastid localization leader peptide recA1 as described above, the primer pair comprising a forward primer and a reverse primer, the forward primer RecA1-F1 is 5'-cgccccgcggaaagcttaattccaatccca-3', as shown in SEQ ID NO.3; the reverse primer RecA1-R1 is 5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3', as shown in SEQ ID NO.4.

[0028] The present application also discloses a transgenic method for delivering nematode-killing proteins to plastids, the method comprising localizing the anti-nematode protein for inhibiting root-knot nematodes in plant plastids through Agrobacterium-mediated nuclear transformation technology combined with plastid localization guide peptide recA1.

[0029] In some embodiments, the key gene for inhibiting root-knot nematodes includes the Cry5Ba2 gene.

[0030] In some embodiments, the method for delivering a nematicidal protein transgene comprises the following steps:

[0031] (1) Construction of expression vector:

[0032] Using the RecA1 fragment as a template, PCR amplification was performed using a primer pair to obtain a recA1 sequence containing overlapping arms of double caMV35s promoter sequences, which was named fragment 4. In the primer pair, the forward primer SOE-RECA-F2 was 5'-ttcatttggagaggacacggatggattcacagctagtct-3', as shown in SEQ ID NO.5; the reverse primer SOE-RECA-R2 was 5'-gctcacCATtctagaatcgaattcagaactgattttgtgg-3', as shown in SEQ ID NO.6;

[0033] Using pWY1 as a template, fragment 4 was amplified by a primer pair to obtain fragment 5, wherein the forward primer SOE-RecA1-F1 is 5'-cgccccgcggaaagcttaattccaatccca-3', as shown in SEQ ID NO.7; the reverse primer SOE-RECA-R1 is 5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3', as shown in SEQ ID NO.8;

[0034] Using fragment 4 and fragment 5 as templates, overlap extension PCR was used to amplify with primers SOE-RecA1-F1 and SOE-RECA-R2, and the resulting fragments were spliced ​​together and named fragment 6;

[0035] (2) Screening and verification of expression vectors:

[0036] Fragment 6 was double-digested with restriction endonucleases SacII and XbaI to recover the target fragment, which was named fragment 7;

[0037] The pWY1 plasmid DNA was double-digested with restriction endonucleases SacII and XbaI to recover the linear vector backbone, which was named fragment 8;

[0038] Fragment 7 and fragment 8 were mixed in an appropriate molar ratio, connected with Solution I enzyme, and cultured. The recombinant was preliminarily identified by enzyme digestion verification, and finally the recombinant was verified by sequencing. The verified recombinant was named pWY24;

[0039] (3) Cloning and expression of expression vector:

[0040] The plasmid pWY24 was transformed into Agrobacterium EHA105 competent cells for cultivation to obtain bacterial liquid;

[0041] The bacterial solution was used as an overlap extension PCR template, and PCR positive clones were selected for restriction digestion and sequencing verification. The correct single clones were screened and the correct strains were stored at -80°C for future use.

[0042] In some embodiments, the preparation of pWY1 specifically comprises the following steps:

[0043] (1) pHW25 plasmid DNA was double-digested with restriction endonucleases XhoI and BamHI, and a linear vector skeleton was recovered. The linear vector skeleton was named fragment 1. The pHW25 plasmid was a pHW25 plasmid containing a double caMV35s promoter and having kanamycin resistance;

[0044] (2) preparing the Cry5Ba2 sequence by PCR amplification using a primer pair, wherein the forward primer Cry5Ba2F is 5'-aagggatccATGGCTACCATCAACG-3', as shown in SEQ ID NO.9; and the reverse primer Cry5Ba2R is 5'-aaagtcgacTCACTGGATCTTGGGC-3', as shown in SEQ ID NO.10;

[0045] (3) The Cry5Ba2 sequence was double-digested with XhoI and BamHI to recover the target fragment, which was named fragment 2;

[0046] (4) Fragment 1 and fragment 2 were mixed in an appropriate molar ratio, ligated with the vector pHW25 under the action of Solution I enzyme, transformed into E. coli XL10-gold for culture, and the recombinant was preliminarily identified by enzyme digestion verification. Finally, the recombinant was verified by sequencing, and the verified recombinant was named pWY1;

[0047] (5) The plasmid pWY1 was transformed into Agrobacterium EHA105 competent cells for cultivation. The obtained bacterial solution was directly used as a PCR template, and PCR amplification was performed for detection using a primer pair. Among the primer pair, the forward primer pWY1agr-F was 5'-cctcctcggattccattgcc-3', as shown in SEQ ID NO.11; the forward primer pWY1agr-R was 5'-GAGTGGAGAGGTTGTAGGAGAGGAA-3', as shown in SEQ ID NO.12.

[0048] In some embodiments, the method for delivering a nematicidal protein transgene uses the freeze-thaw method to introduce a plasmid containing the target gene into Agrobacterium.

[0049] The present application also discloses an application of a nematicidal protein delivery transgene obtained by the above method in the preparation of root-knot nematode resistant plants.

[0050] In some embodiments, plasmid pHW25 is used as an expression vector to construct a vector expressing the Cry5Ba2 gene, and the vector is transformed into plant leaves to cultivate plants resistant to root-knot nematodes.

[0051] In some embodiments, the plant comprises tobacco.

[0052] The following is an explanation through specific embodiments.

[0053] In the following embodiments of the present application, when the temperature is not specifically limited, the normal temperature condition is used. Normal temperature refers to the natural greenhouse condition in all seasons without additional cooling or heating treatment, and is generally controlled at 10-30°C, preferably 15-25°C.

[0054] The genes, proteins or fragments thereof involved in the present application may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants, etc.) using recombinant technology.

[0055] This application is based on the synthesis of the Bt gene Cry5Ba2, which has been reported to have resistance to root-knot nematodes, and the leader peptide recA1 has the efficiency of efficiently locating the protein in the root plastid, and designs a plant expression vector, and develops a preparation method of a transgenic plant with plastid-delivered nematicidal protein and its application in resisting root-knot nematodes. First, it was found that root-knot nematodes can feed on the endogenous protein D1 in the plastid, and then a tobacco expression vector was constructed. The efficient expression of Cry5Ba2 in tobacco was successfully detected by Western blot, and tobacco strains with high expression levels were selected, and on this basis, physiological tests of root-knot nematodes were carried out. The specific examples are shown in the following examples.

[0056] The bacterial species involved in the present application include: Agrobacterium tumefaciens EHA105; a double caMV35s promoter and a kanamycin resistance plasmid pHW25 expression vector, which is used as a plant transformation vector for expressing cytoplasmic-localized and plastid-localized Cry5Ba2.

[0057] 1. Verification of nematode feeding on plastid proteins

[0058] 1.1 Reagent preparation

[0059] Nematode protein extraction lysis buffer:

[0060] Table 1

[0061]

[0062] 1.2 Root-knot nematode inoculation

[0063] The second-instar larvae of the infective southern root-knot nematode were inoculated around the roots of the tobacco strains expressing GFP and wild-type, and infected for 35-45 days. The plant growth greenhouse conditions were 25℃ / 20℃light / dark 16h / 8h.

[0064] 1.3 Isolation of female root-knot nematodes

[0065] 1) After 35-45 days, pull up the tobacco roots and clean the soil around the roots.

[0066] 2) Cut the roots into 2-3cm segments, then put them into a mortar and pound them to break the root knots.

[0067] 3) Re-spin with ddH2O, then sieve through 18 mesh, 60 mesh, 100 mesh, 200 mesh, 300 mesh, and 500 mesh in sequence. Female root-knot nematodes are in 60 mesh, J3 and J4 are in 200 mesh and 300 mesh, and eggs are in 500 mesh.

[0068] 4) Rinse the female worms on the 60-mesh plate with ddH2O and collect them in a plate.

[0069] 5) Under a dissecting microscope, use a 200uL pipette (with the tip cut off) to transfer the female worms to a new dish containing ddH2O.

[0070] 6) Wash the female insects 3-4 times with sterile ddH2O to clean the plant tissue.

[0071] 7) The female insects were placed into sterile EP tubes and stored at -80°C for later use.

[0072] 1.4 Western blot of female root-knot nematodes

[0073] 1) The female root-knot nematodes separated above were added with an appropriate amount of nematode protein extraction and lysis solution, and the mixture was repeatedly frozen and thawed twice with liquid nitrogen.

[0074] 2) Add 2 zirconium oxide strains, freeze quickly in liquid nitrogen, grind in a proofing machine, soak in liquid nitrogen first, lay out symmetrically, 55HZ 40S, grind thoroughly to prevent thawing.

[0075] 3) Repeat the grinding twice to fully lyse the nematodes.

[0076] 4) Centrifuge at 12000 rpm for 10 min at 4°C.

[0077] 5) Add the same volume of protein loading buffer and heat at 100°C for 5 min to prepare female insect protein. Use western blot to detect protein expression (D1-specific antibody). Figure 1 shown.

[0078] 2. Acquisition of Cry5Ba2 gene

[0079] The Cry5Ba2 gene was synthesized by a third-party company, Jinkairui Biological Co., Ltd., and the sequence is shown in SEQ ID NO.1.

[0080] 3. Obtaining Agrobacterium Competent Cells

[0081] 1. Preparation of Agrobacterium Competent Cells

[0082] (1) Activate Agrobacterium tumefaciens EHA105 by streaking, pick a single colony of Agrobacterium tumefaciens EHA105 in 5 ml of YEB liquid medium (containing 50 mg / l Rif), and culture with shaking at 28°C overnight;

[0083] (2) Take 1 ml of overnight culture solution and inoculate it into 50 ml YEB (Rif 50 mg / l) liquid medium, and culture it at 28°C with shaking until the OD600 reaches 0.5;

[0084] (3) Divide the bacterial solution into two 50 mL centrifuge tubes (balanced), centrifuge at 5000-6000 rpm for 5 min (or 4000 rpm for 10 min), and discard the supernatant;

[0085] (4) Add 12.5 ml (1 / 2 of the bacterial solution) of 20 mM CaCl2 to each tube to fully suspend the Agrobacterium cells and place on ice for 30 min.

[0086] (5) Centrifuge at 5000-6000 rpm for 5 min (or 4000 rpm for 10 min), discard the supernatant, place on ice, add 6.25 mL (1 / 4 of the bacterial solution) of pre-cooled 20 mM CaCl2, fully suspend the cells, and dispense 100-200 μL per tube into 1.5 ml EP tubes. Quick-freeze in liquid nitrogen for 1 min and store in a -80°C refrigerator for later use.

[0087] 4. Construction of plant expression vector

[0088] In this example, the plasmid pHW25 containing double caMV35s promoters and kanamycin resistance was used as an expression vector and introduced into Agrobacterium tumefaciens EHA105 as a plant transformation vector for expressing cytoplasmic-localized and plastid-localized Cry5Ba2 and GFP proteins.

[0089] Plasmid pHW25 was used as the backbone, and the synthetic Cry5Ba2 gene (sequence shown in SEQ ID NO.1) and the plastid localization peptide were used as recA1 from Arabidopsis thaliana (sequence shown in SEQ ID NO.2) to construct a series of plant expression vectors: pHW25+nuCry5Ba2; pHW25+nuTPCry5Ba2

[0090] Construction of pHW25+nuCry5Ba2(WY1) plant expression vector: First, pHW25 plasmid DNA was double-digested with restriction endonucleases XhoI and BamHI at 37°C for 3 hours, and the linear vector backbone was recovered, and this fragment was named fragment 1. The restriction digestion system was as follows: pHW25: 10.0μL; XhoI and BamHI 0.5μL each; 10×buffer 5.0μL; ddH2O 34.0μL. A 2098bp Cry5Ba2 protein sequence was synthesized at a third-party company, and primers Cry5Ba2F (5'-aagggatccATGGCTACCATCAACG-3') and Cry5Ba2R (5'-aaagtcgacTCACTGGATCTTGGGC-3') were designed for amplification. The PCR reaction system was as follows:

[0091] Table 2

[0092] Components Usage 10μM Cry5Ba2 F 1μL 10 μM Cry5Ba2 R 1μL template 1μL 2×Taq Plus Master Mix 15μL ddH2O To 30μL

[0093] The fragment with XhoI at the front and BamHI at the back was obtained. The target fragment was recovered after double digestion with XhoI and BamHI and named as fragment 2. Fragments 1 and 2 were mixed in a molar ratio of 1:3-10 and connected with the vector pHW25 at 16°C. The system was as follows:

[0094] Table 3

[0095] Components Usage Restriction vector backbone 1 2μL Target fragment 2 3μL Solution I 5μL

[0096] The specific volume of the target fragment in the system is calculated according to the sample concentration so that the sample dosage meets the molar ratio. After ligation, the E. coli XL10-gold was transformed and screened on a plate containing kanamycin. The recombinant was preliminarily identified by enzyme digestion verification, and finally the recombinant was verified by sequencing. The verified recombinant was named pWY1. The results are as follows Figure 2 shown.

[0097] The plasmid was then transformed into Agrobacterium EHA105 competent cells and spread on LB medium with kanamycin added. After 12 hours, single colonies were randomly picked from the medium and inoculated into 50 mg / L LB liquid medium containing kanamycin. They were cultured overnight (37°C, 220 rpm) and the cultured bacterial solution was directly used as a PCR template for detection.

[0098] The method of introducing plasmid DNA into Agrobacterium is as follows:

[0099] Freeze-thaw method:

[0100] 1. Add 2-10 μL (about 1-10 μg) plasmid DNA to 200 μL competent cells, place on ice for 5 minutes, and freeze in liquid nitrogen for 5 minutes;

[0101] 2. Rapidly transfer to a 37°C water bath and heat shock for 5 minutes;

[0102] 3. Add 1 mL YEB (without antibiotics) liquid culture medium and culture at 28°C with shaking at 180-200 rpm for 2-4 hours;

[0103] 4. Centrifuge at 3000 rpm for 4 min, remove part of the supernatant, and use 200 μL of the bacterial solution to spread on a YEB plate containing 50 mg / L Kan and 50 mg / L Rif;

[0104] 5. Leave it for about 0.5 hours, wait for the water to dry up, and then culture it at 28℃ for about 24 hours until colonies grow.

[0105] 6. The grown colonies were directly used as PCR templates and detected using primers pWY1agr-F (5'-cctcctcggattccattgcc-3', as shown in SEQ ID NO.11) and pWY1agr-R (5'-GAGTGGAGAGGTTGTAGGAGAGGAA-3', as shown in SEQ ID NO.12).

[0106] The colony PCR reaction system is as follows:

[0107] Table 4

[0108]

[0109] After mixing, PCR reaction was performed: 95°C, 5 min; 95°C, 30 s, 56°C, 30 s, 72°C, 30 s, 35 cycles; 72°C, 5 min. PCR positive clones were selected for sequencing verification, and the correct single clones were screened. The strains with correct sequencing were stored at -80°C for future use.

[0110] Construction of pHW25+nuTPCry5Ba2(WY24) plant expression vector: Using Arabidopsis cDNA as template, primers RecA1-F1 (5'-cgccccgcggaaagcttaattccaatccca-3') and RecA1-R1 (5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3') were designed to amplify a 195 bp recA1 sequence (the PCR system was the same as before), and this fragment was named fragment 3. The obtained RecA1 fragment was used as a template, and primers SOE-RECA-F2 (5'-ttcatttggagaggacacggatggattcacagctagtct-3') and SOE-RECA-R2 (5'-gctcacCATtctagaatcgaattcagaactgattttgtgg-3') were designed to obtain the recA1 sequence containing overlapping arms of the double caMV35s promoter sequence (PCR system is the same as before), and this fragment was named fragment 4. Using the constructed pWY1 as a template, primers SOE-RecA1-F1 (5'-cgccccgcggaaagcttaattccaatccca-3') and SOE-RECA-R1 (5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3') were designed to amplify the double caMV35s promoter sequence containing overlapping arms of the recA1 sequence (PCR system is the same as before), and this fragment was named fragment 5. Using fragment 4 and fragment 5 as templates, overlap extension PCR was used to amplify and splice them with the two pairs of primers SOE-RecA1-F1 and SOE-RECA-R2.

[0111] The system and procedure of overlap extension PCR are as follows:

[0112] Table 5

[0113] Components Usage 2×TSINGKE Master Mix 15μL 10 μM pWY1agr-F 1μL 10μM pWY1agr-R 1μL template 4, 5 0.5 μL each ddH2O 4.0μL

[0114] After mixing, PCR reaction was carried out: 95°C, 3 min; 95°C, 15 s, 55°C, 15 s, 72°C, 30 s (1 min / kb), 35 cycles; 72°C, 5 min, sample loading, electrophoresis, observation of target bands in a UV gel imaging system, and recovery of target fragment 6.

[0115] The obtained target fragment 6 was double-digested with restriction endonucleases SacII and XbaI at 37°C, and the target fragment was recovered and named fragment 7 (the enzyme digestion system was the same as before). After double-digesting the pWY1 plasmid DNA with restriction endonucleases SacII and XbaI at 37°C for 3 hours, the linear vector skeleton was recovered and this fragment was named fragment 8. Fragments 7 and 8 were mixed at a molar ratio of 10:1, connected with Solution I enzyme, and transformed into Escherichia coli XL10-gold, screened on a plate containing kanamycin, and the recombinant was preliminarily identified by enzyme digestion verification. Finally, the recombinant was verified by sequencing, and the verified recombinant was named pWY24. The results are as follows Figure 2 shown.

[0116] Then the plasmid was transformed into Agrobacterium EHA105 competent cells and spread on YEB medium with kanamycin. After 12 hours, single colonies were randomly picked from the medium and inoculated into 50 mg / L YEB liquid medium containing kanamycin, and cultured overnight (37°C, 220rpm). The cultured bacterial liquid was directly used as a PCR template. The PCR reaction system was the same as above, and PCR positive clones were selected for enzyme digestion and sequencing verification. The correct single clone was screened and the correct strains were stored at -80°C for future use.

[0117] 5. Preparation and identification of plants transformed with nuclear genome

[0118] 1. Preparation of reagents and culture medium

[0119]

[0120] After sterilization, add per 500 ml of culture medium:

[0121] 0.1mg / L NAA 1mg / ml NAA 50μL

[0122] 1mg / L 6-BA 1mg / ml6-BA 500μL

[0123] YEB:

[0124]

[0125] pH 7.0, autoclaved.

[0126] Solid culture medium:

[0127] Add 15 g agar powder per liter of YEB liquid medium and sterilize by high pressure.

[0128] Kanamycin (Kan) stock solution: 100 mg / ml

[0129] Rifampicin (Rif) stock solution: 50mg / ml

[0130] YEB solid culture medium plate: When the temperature of the sterilized YEB solid culture medium drops to 50°C, add kanamycin (Kan) and rifampicin (Rif) to a final concentration of 50 mg / l, mix well and immediately pour into a culture dish. After solidification, store inverted at 4°C.

[0131] 2. Agrobacterium-mediated transformation of tobacco cell nuclear genome

[0132] (1) Streak Agrobacterium on a plate, pick a single colony from the plate, and inoculate it into 20 mL of YEB liquid medium supplemented with corresponding antibiotics (kan 50 mg / L, Rif 50 mg / L). Cultivate on a constant temperature shaker at 28°C and 180 r / min until OD600 reaches 0.6-0.8 (about 17 h).

[0133] (2) Transfer the bacterial solution with an OD600 of 0.6 to 0.8 into a newly prepared YEB liquid medium without antibiotics (containing Rif) at a ratio of 1% to 2%, and add 100 to 500 μmol / L of acetosyringone. Cultivate for another 6 hours under the same conditions as above, and when the OD600 is 0.2 to 0.5, it can be used for transformation.

[0134] (3) Centrifuge at 4°C and 2500 r / min to precipitate the bacteria. Pour the bacterial solution into a glass plate on a clean bench. Place tobacco leaves (about one month old) cut into 5×5 mm in size into the bacterial solution and soak for 5-10 min (usually about 10 min). Remove the leaves and place them on sterile filter paper to absorb the attached bacterial solution.

[0135] (4) Place the infected leaves on RMOP and culture them in the dark at 28°C for 2 days (until a circle of Agrobacterium grows around them).

[0136] (5) The co-cultivated explants were transferred to RMOP (with 200 mg / L Tim added to inhibit the growth of Agrobacterium tumefaciens) with antibiotics (100 mg / L kana) and selectively cultured at 25°C at an illumination of 25 μmol·m-2·s-1.

[0137] (6) After 2-3 weeks of selective culture, the transformed cells of the explants will differentiate into resistant adventitious buds or produce resistant callus. These resistant materials are transferred to the corresponding selective culture medium (RMOP+100mg / L kana+200mg / L Tim) for subculture.

[0138] (7) When the adventitious buds grow to more than 1 cm, cut them off and insert them into tobacco rooting medium containing selective pressure (MS+0.1mg / LNAA+100mg / L kana+200mg / L Tim) for rooting culture. Adventitious roots will grow in about two weeks and can be used for molecular identification after they grow a little bigger.

[0139] 3. Identify positive nuclear transfection plants by PCR

[0140] 3.1 Reagent preparation:

[0141] CTAB-Extraction Buffer(100ml):

[0142]

[0143] Add β-ME (mercaptoethanol) at the final use and dilute at a ratio of 2:100 (add 2 ml β-ME to 100 ml buffer).

[0144] 3.2 CATB method for DNA extraction

[0145] 1. Take about 100 mg of plant leaves (the size of a thumb cap) and place them in a 2 ml EP tube equipped with three sterilized steel balls. Quickly freeze them in liquid nitrogen and grind them in a proofer. Soak them in liquid nitrogen first, place them symmetrically, and grind them at 55HZ for 40S to prevent thawing.

[0146] 2. Add 500 μl CTAB extraction solution, vortex for 30 seconds, warm at 60°C for 30 minutes (mix every ten minutes).

[0147] 3. Add 200 μl of chloroform / isoamyl alcohol (24:1) mixture and mix until milky white.

[0148] Centrifuge at 4.4℃, 12000rpm, 10min, take the supernatant into a new 1.5ml EP tube,

[0149] Add 0.8-0.9 volume of isopropanol and mix by inversion.

[0150] Centrifuge at 5.4°C, 12000 rpm, for 30 min.

[0151] 6. Discard the supernatant and add 70% ethanol to wash the precipitate (0.5-1 ml) 1-2 times.

[0152] 7. Microcentrifuge, discard the supernatant, dry naturally, add about 40μl ddH2O to dissolve, measure the concentration, and store at -20℃.

[0153] 3.3 PCR identification of positive plants

[0154] The extracted plant DNA was used as a template and primers pWY1agr-F and pWY1agr-R were used to identify the transgenic plant WY1. The PCR reaction system was as follows:

[0155]

[0156]

[0157] After mixing, PCR reaction was carried out: 95°C, 5 min; 95°C, 30 s, 56°C, 30 s, 72°C, 30 s, 35 cycles; 72°C, 5 min.

[0158] The extracted plant DNA was used as a template and primers pWY1agr-F and pWY1agr-R were used to identify the transgenic plant WY24. The PCR reaction system was the same as above.

[0159] 6. Plant expression, detection and comparison of Cry5Ba2 protein

[0160] 5.1 Reagent preparation:

[0161] Extraction buffer (500mL)

[0162]

[0163] Adjust pH to 9.4 with KOH and make up to 500 mL. Before use, add 2% β-mercaptoethanol (10 μL) 5 μM and protease inhibitors (100 mM, 10 μL) to every 500 μL;

[0164] NH4Ac in methanol (500mL): 0.1M is 3.854g NH4Ac dissolved in 500mL methanol.

[0165] 5.2 The operation steps are as follows:

[0166] 1) Take about 200 mg of tobacco plant roots and freeze them quickly in liquid nitrogen, grind them in a proofer, soak them in liquid nitrogen first, lay them out symmetrically, grind them thoroughly at 55HZ for 40S.

[0167] 2) Add 500 μL extraction buffer to the plant material and vortex for 30 seconds.

[0168] 3) Add 500 μL of phenol, vortex for 30 s, 4°C, 12000 rpm, 10 min.

[0169] 4) Transfer the supernatant to a new EP tube.

[0170] 5) Add 1 mL of 0.1 M NH4Ac per 200 μL of extract and vortex for 10 seconds.

[0171] 6) Place in -20℃ refrigerator to precipitate protein.

[0172] 7) 12000rpm, 4℃, 5min.

[0173] 8) Wash twice with 500 μL 0.1 M NH4Ac methanol solution and dry at room temperature.

[0174] 9) Dissolve with 30-100 μL of 1% SDS.

[0175] 10) Centrifuge at 12000 rpm, 4°C for 3 min, aspirate the supernatant and use Nano photometer to measure the approximate protein concentration range, and then dilute to a concentration of 0.5 mg / mL.

[0176] 11) Use the BCA kit to quantify the protein.

[0177] 12) Resuspend the protein solution with 1% SDS solution and dilute it with protein loading buffer to a loading solution of 1 ug / uL, heat it at 100°C for 5 min to prepare a protein sample, and detect the protein expression by western blot (Cry5Ba2-specific antibody).

[0178] Results Figure 3 As shown, Cry5Ba2 protein with a size of about 79 kDa was accurately detected in pHW25+nuCry5Ba2 and pHW25+nuTPCry5Ba2, indicating that the induced expression of the plant was successful. Then, the WY1#5, 10 and WY24#10, 15 strains with the highest and similar expression levels were selected for bioassay experiments.

[0179] 7. Determination of the effect of plant expression vector against southern root-knot nematode

[0180] After the tobacco seeds of this example germinated in the soil, they were transplanted into a pot with a diameter of 11 cm and a height of 9 cm and filled with sand and soil in a ratio of 1:1. After they grew to the size of 4-5 true leaves, the plants were dug out and 500 J2s / pot were inoculated around the rhizosphere soil. After 35 days, the number of root knots and eggs of different plants was counted. The greenhouse conditions were 25℃ / 20℃light / dark 16h / 8h.

[0181] The number of root nodes in transgenic plants with plastid-delivered Cry5Ba2 was significantly lower than that in the control group (e.g. Figure 4 , significance analyzed by Tukey HSD).

[0182] In addition, the number of eggs in transgenic plants with plastid-delivered Cry5Ba2 was significantly lower than that in the control group ( Figure 5 ). The above results show that the plastid delivery of anti-nematode protein technology can improve the resistance of plants to root-knot nematodes, reduce the number of root knots of root-knot nematodes and inhibit the number of offspring.

[0183] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A gene for a plastid localization leader peptide recA1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

2. A method for delivering a nematicidal protein transgene, the method comprising recombinantly expressing the Cry5Ba2 gene that inhibits root-knot nematodes in tobacco through Agrobacterium-mediated nuclear transformation technology combined with a plastid-localized leader peptide recA1, wherein the nucleotide sequence of the Cry5Ba2 gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the plastid-localized leader peptide recA1 gene is shown in SEQ ID NO.

2.

3. The method according to claim 2, comprising the following steps: (1) Construction of expression vector: Using SEQ ID NO.2 as a template, PCR amplification was performed using SOE-RECA-F2 and SOE-RECA-R2 as a primer pair to obtain fragment 4 containing overlapping arms of double caMV35s promoter sequences, wherein SOE-RECA-F2 is 5'-ttcatttggagaggacacggatggattcacagctagtct-3'; SOE-RECA-R2 is 5'-gctcacCATtctagaatcgaattcagaactgattttgtgg-3'; Using pWY1 plasmid as template, PCR amplification was performed using SOE-RecA1-F1 and SOE-RECA-R1 as primer pairs to obtain fragment 5, wherein SOE-RecA1-F1 is 5'-cgccccgcggaaagcttaattccaatccca-3'; the reverse primer SOE-RECA-R1 is 5'-agactagctgtgaatccatccgtgtcctctccaaatgaa-3'; Using the fragment 4 and the fragment 5 as templates and SOE-RecA1-F1 and SOE-RECA-R2 as primer pairs, overlap extension PCR was performed to amplify and obtain fragment 6; (2) Screening and verification of expression vectors: The fragment 6 is double-digested with restriction endonucleases SacII and XbaI to recover the target fragment to obtain fragment 7; The pWY1 plasmid was double-digested with restriction endonucleases SacII and XbaI, and the linear vector backbone was recovered to obtain fragment 8; The fragment 7 and the fragment 8 were mixed at a molar ratio of 10:1, and the mixture was cultured after being connected with Solution I enzyme, and the recombinant was preliminarily identified by enzyme digestion verification, and the recombinant was verified by sequencing, and the pWY24 plasmid was extracted from the verified correct recombinant; (3) Cloning and expression of expression vector: The pWY24 plasmid is transformed into Agrobacterium EHA105 competent cells for cultivation to obtain bacterial liquid; The bacterial solution was used as an overlap extension PCR template, and PCR positive clones were selected for restriction digestion and sequencing verification to screen out the correct single clones. The correct strains sequenced were stored at -80°C for future use.

4. The method according to claim 3, wherein the preparation of pWY1 specifically comprises the following steps: (1) The pHW25 plasmid was double-digested with restriction endonucleases XhoI and BamHI, and the linear vector backbone was recovered to obtain fragment 1. The pHW25 plasmid contained a double caMV35s promoter and a kanamycin resistance gene; (2) preparing the Cry5Ba2 sequence by PCR amplification using Cry5Ba2F and Cry5Ba2R as a primer pair, wherein Cry5Ba2F is 5'-aagggatccATGGCTACCATCAACG-3', and Cry5Ba2R is 5'-aaagtcgacTCACTGGATCTTGGGC-3'; (3) double digesting the Cry5Ba2 sequence with XhoI and BamHI to recover the target fragment, which is fragment 2; (4) Fragment 1 and Fragment 2 are mixed at a molar ratio of 1:3-10, and ligated under the action of Solution I enzyme. The ligated product is transformed into Escherichia coli XL10-gold for culture, and the recombinant is preliminarily identified by enzyme digestion verification. Finally, the recombinant is verified by sequencing, and the pWY1 plasmid is extracted from the verified recombinant; (5) The pWY1 plasmid was transformed into Agrobacterium EHA105 competent cells for cultivation. The obtained bacterial solution was directly used as a PCR template. PCR amplification detection was performed using pWY1agr-F and pWY1agr-R as a primer pair. The pWY1agr-F was 5'-cctcctcggattccattgcc-3', and the pWY1agr-R was 5'-GAGTGGAGAGGTTGTAGGAGAGGAA-3'. 5 . The method for delivering a nematicidal protein transgene according to claim 4 , wherein the pWY1 plasmid is introduced into the Agrobacterium EHA105 using a freeze-thaw method.

6. Use of a transgenic plant delivering nematicidal protein obtained by the method of any one of claims 2 to 5 in the preparation of root-knot nematode-resistant tobacco.

7. The use according to claim 6, wherein the pHW25 plasmid is used as an expression vector to construct a vector expressing the Cry5Ba2 gene, and the vector is transformed into tobacco leaves to culture tobacco resistant to root-knot nematodes.

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