Fusion protein for preventing and treating cowpea resistant lepidoptera pests and application

Abstract

The invention discloses a fusion protein for preventing and treating cowpea resistant lepidoptera pests and application thereof.The fusion protein of Cry1Ab and mutated Vip3Af is screened out, insecticidal proteins Cry and Vip3 in the fusion protein are expressed at the same time in plants, it is beneficial for the insect-resistant protein to exert biological activity, and the effect of preventing and treating cowpea resistant lepidoptera pests is achieved. The risk of occurrence of pest resistance in nature is effectively relieved, and resistant lepidoptera pests are efficiently prevented and treated. Compared with the existing insecticidal protein cowpea, the cowpea for expressing the insecticidal fusion protein provided by the invention has higher insecticidal activity on lepidoptera pests.

Description

(I) Technical field

[0001] The invention relates to a method for preventing and controlling cowpea pests and resistant pests, and in particular to a fusion protein for preventing and controlling cowpea lepidopteran pests and resistant lepidopteran pests and an application thereof. (II) Background technology

[0002] Pests cause billions of dollars in losses to global agricultural production every year. Pest control currently relies mainly on the use of chemical pesticides and the promotion of genetically modified crops. The residues of chemical pesticides can have adverse effects on human health and the environment. Using genetic engineering technology to introduce and integrate genes encoding insect-resistant proteins into the genome of crops to obtain genetically modified crops that can express specific insect-resistant proteins has been a trend in industrial development over the past 30 years.

[0003] Long-term and large-scale use of a single insecticidal protein can lead to the generation and development of pest resistance to BT insecticidal proteins (Carriere, Crickmore et al. 2015). Since the first case of BT-resistant pests was discovered in the world in 2005, the global incidence of resistant pests has increased year by year and is showing a rapid growth trend (Tabashnik and Carrière 2017). Lepidopteran pests resistant to BT proteins have been screened or discovered in both the laboratory (in an artificial climate environment) and the field (in a natural environment). Currently, there are two types of BT insecticidal proteins that are most widely used and have the best insecticidal effects on lepidopteran insects, namely insecticidal crystal proteins (CRYs) and vegetative insecticidal proteins (VIPs). The insecticidal mechanism (mode of action) of Vip proteins is completely different from that of Cry proteins, and the amino acid sequences of these two types of proteins have no homology, and there is no cross-resistance between the two. Vip3A protein and insecticidal crystal protein Cry have different binding receptors in the insect midgut, and their toxicity mechanisms to insects are different, so there is no cross-resistance. Chakroun et al. summarized the literature and pointed out that the typical binding receptors of proteins such as Cry1A and Cry1Fa on the insect midgut cannot interact with Vip3 protein, and the mechanism of action of insecticidal crystal protein and Vip3 protein in the insect midgut is different (Chakroun, Banyuls et al. 2016). From the perspective of insecticidal spectrum, Vip3A protein has high insecticidal activity against Lepidoptera pests of Noctuidae (such as fall armyworm, cotton bollworm, etc.), but has low or no insecticidal activity against Pyralidae pests (such as corn borer) (Chakroun, Banyuls et al. 2016). The most diverse and most commonly used Vip3Aa proteins (such as Vip3Aa1, Vip3Aa20, Vip3Aa45, etc.) have no insecticidal activity against Pyralidae pests (such as corn borer, striped stem borer, etc.). The use of "gene stacking" strategy can effectively manage the occurrence of resistant pests.

[0004] Cowpea is an important vegetable crop and food crop in the world. It is seriously damaged by lepidopteran insects during cultivation. There are also reports on the transfer of Bt genes into cowpea and the cultivation of insect-resistant transgenic cowpea. Australian scholars transferred the gene encoding cry1Ab protein into cowpea plants to achieve the purpose of controlling cowpea pod borer (Popelka et al., 2006). Subsequently, they carried out field tests for many years to verify that the transgenic crop was indeed active against cowpea pod borer and conducted risk assessment on non-target organisms in the field (Ba et al., 2018). Bakshi et al. studied the transformation system of introducing Cry1Ac into cowpea (Bakshi and Sahoo, 2013), but did not report whether the transformant was active against cowpea pod borer. Bett et al. studied the activity of Vip protein against cowpea pod borer and found that Vip3Ba inhibited the growth of its larvae and introduced the gene into cowpea (Bett et al., 2017). Recently, Kumar et al. reported that Cry2Aa-transformed cowpea had a mortality rate of more than 90% against cowpea pod borer larvae (Kumar et al., 2021). In January 2019, the cowpea transformation event AAT709A was commercialized in Nigeria (www.isaaa.org). All of the above express a single insecticidal protein, which is easy to cause cowpea pests to develop resistance to it.

[0005] The present application conducts detailed biological activity assays on cowpea pests, especially important Lepidoptera pests such as resistant cowpea pod borer that are insensitive to Cry proteins, and screens out the insecticidal activity of the fusion protein of Cry1Ab and the mutated Vip3Af against cowpea pod borer and resistant pests. It is found that the fusion insecticidal protein has a synergistic insecticidal effect on cowpea pests, and has high application value and broad application prospects. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a fusion protein for controlling resistant lepidopteran pests of cowpea and its application. The present invention adopts a strategy of fusing Cry protein and mutated Vip protein to achieve the purpose of controlling cowpea pests, especially important cowpea pests such as resistant cowpea pod borer that are insensitive to Cry protein, while delaying the occurrence of pest resistance to Bt protein.

[0007] The technical solution adopted by the present invention is:

[0008] The invention provides a fusion protein for preventing and controlling resistant lepidopteran pests of cowpea. The fusion protein is formed by fusion connection of a BT insecticidal crystal protein and a BT vegetative stage insecticidal protein. The BT insecticidal crystal protein is a BT insecticidal crystal protein Cry1Ab; the BT vegetative stage insecticidal protein is a mutated BT vegetative stage insecticidal protein Vip3Af, and the nucleotide sequence is shown in SEQ ID NO:2.

[0009] Furthermore, the BT insecticidal crystal protein Cry1Ab has a nucleotide sequence as shown in SEQ ID NO:1.

[0010] Furthermore, the nucleotide sequence of the fusion protein is shown in SEQ ID NO:3.

[0011] The invention provides a recombinant DNA molecule of the fusion protein, which comprises a nucleotide sequence encoding the fusion protein and a promoter sequence for starting the expression of the fusion protein in plants.

[0012] Further, the promoter includes a bacterial promoter or a plant promoter. Plant promoters include inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and / or spatiotemporally regulated promoters known in the art.

[0013] The present invention provides a DNA construct comprising the recombinant DNA molecule, which exists in the genome of a transgenic plant, seed or cell.

[0014] The present invention also provides a method for controlling cowpea lepidopteran pests and resistant lepidopteran pests using the fusion protein, the method comprising: contacting the fusion protein with the target pest, or transferring the fusion protein into the plant genome, so as to achieve the purpose of controlling the target pest.

[0015] Further, the target pests include Lepidoptera pests or resistant Lepidoptera pests, the Lepidoptera pests include cowpea pod borer, and the resistant Lepidoptera pests include resistant cowpea pod borer that is insensitive to Cry toxins.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] (1) The present invention screened out a fusion protein of Cry1Ab and the mutated Vip3Af. The insecticidal proteins Cry and Vip3 in the fusion protein were simultaneously expressed in plants, which is beneficial to the biological activity of the insect-resistant protein. It not only effectively reduces the risk of pest resistance in nature, but also effectively prevents and controls resistant Lepidoptera pests.

[0018] (2) Compared with the existing insecticidal protein cowpea, the cowpea expressing the insecticidal fusion protein provided by the present invention has higher insecticidal activity against lepidopteran pests. (IV) Specific implementation methods

[0019] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0020] The molecular biology and biochemical methods used in the following examples of the present invention are all known techniques. Detailed descriptions are provided in documents such as Current Protocols in Molecular Biology, compiled by Ausubel and published by John Wiley and Sons, and Molecular Cloning: A Laboratory Manual, 3rd ED., compiled by J. Sambrook et al. and published by Cold Spring Harbor Laboratory Press (2001).

[0021] Example 1. Construction of insect-resistant fusion gene vector and preparation of recombinant protein

[0022] The coding genes involved in the present invention are all artificially synthesized by Shanghai Shenggong Company, wherein the insect-resistant fusion protein Cry1Ab-Vip3Af is composed of BT insecticidal crystal protein Cry1Ab and mutated BT vegetative insecticidal protein Vip3Af through fusion connection, and the nucleotide sequence of the coding gene cry1ab-vip3af is SEQ ID NO: 3. The nucleotide sequence of BT insecticidal crystal protein Cry1Ab is SEQ ID NO: 1; the nucleotide sequence of mutated BT vegetative insecticidal protein Vip3Af is SEQ ID NO: 2.

[0023] The Escherichia coli pET expression system is used to express and prepare recombinant proteins, that is, the target gene is introduced into a suitable expression vector, and the expression vector is then transferred into a suitable expression strain. The expression of the target gene is induced under certain conditions to prepare the recombinant protein.

[0024] The preparation process of the insect-resistant fusion protein Cry1Ab-Vip3Af is as follows: the fusion gene cry1ab-vip3af is cloned between the two restriction endonuclease sites of BamHI and SacI of the expression vector pET32a (Novagen, 69015-3, USA) to obtain the recombinant plasmid pET32a-cry1ab-vip3af; the recombinant plasmid is introduced into the Escherichia coli BL21 (DE3) cell line by heat shock transformation method, and cultured at 37°C overnight on LB solid medium containing 50 mg / L kanamycin to screen for monoclonal positive colonies; a single colony is inoculated into LB liquid medium containing 50 mg / L ampicillin, and after overnight culture at 37°C, 0.2 ml of the bacterial solution is subcultured and inoculated into a conical flask containing 200 ml of LB liquid culture medium containing 50 mg / L ampicillin, and shaken and cultured at 37°C until OD600 = 0.6, and then added IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and the shaking culture was continued under the same conditions for 4 hours to induce the expression of the recombinant protein. After the expression was completed, the bacterial culture was centrifuged at 5000g for 15 minutes, the supernatant culture was discarded, the cell pellet was collected, and the cells were resuspended with 50 ml of 20 mM phosphate buffer (PBS, pH = 7.4); the resuspended solution was then placed in ice water for sufficient cooling, and the cells were crushed with an ultrasonic crusher (probe diameter 10 mm, crushed at 650W, 20KHz for 15 minutes), centrifuged, and the supernatant and the precipitate were identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the protein sample containing the correct target band was stored in a -80°C refrigerator for use, and the anti-insect fusion protein Cry1Ab-Vip3Af suspension was obtained, which was the recombinant protein suspension. The LB liquid culture medium is composed of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, the solvent is double distilled water, and it is sterilized at 120°C and high temperature and high pressure for 15 minutes; the LB solid culture medium is prepared by adding 15 g / L agar to the LB liquid culture medium, sterilizing it at high temperature and high pressure, and then inverting it in a sterilized culture dish to cool it.

[0025] The above is a method for preparing the fusion protein Cry1Ab-Vip3Af. In the same experimental method, insecticide proteins Cry1Ab and Vip3Af can be obtained. At the same time, this example also prepared a fusion protein of Cry1Ab and unmutated Vip3Af (nucleotide sequence is SEQ ID NO: 4) as a control for subsequent examples.

[0026] Example 2: Determination of the biological activity of the recombinant protein against cowpea lepidopteran insects

[0027] 1. Experimental subjects: Cowpea pod borer and resistant cowpea pod borer insensitive to Cry1Ab toxin were selected as test insects. Resistant cowpea pod borer was collected from the Sanya Experimental Base in Hainan Province and obtained through artificial breeding screening. The specific method was based on the method provided in the literature (Kain, Wendy C., et al. "Inheritance of resistance to Bacillus thuringiensis Cry1Ac toxin in a greenhouse-derived strain of cabbage looper (Lepidoptera: Noctuidae)." Journal of economic entomology 97.6 (2004): 2073-2078.; Huang, Fangneng, et al. "Baseline susceptibility and changes in susceptibility to Bacillus thuringiensis subsp. kurstaki under selection pressure in European corn borer (Lepidoptera: Pyralidae)." Journal of Economic Entomology 90.5 (1997): 1137-1143.). The resistant cowpea pod borer is insensitive to Bt insecticidal crystal protein (Cry1Ab), while the common cowpea pod borer is sensitive to Bt insecticidal crystal protein.

[0028] 2. Experimental group: The recombinant protein prepared by the method of Example 1 was subjected to a preliminary test of biological activity (1 ng / cm 2 、10ng / cm 2 , 100ng / cm 2 、1000ng / cm 2 Four groups of concentrations); according to the preliminary experimental results, 8 groups of protein concentration gradients (the concentration range is shown in Table 1 below, diluted with PBS solution) were set to determine the concentration range with 50% lethality to insects. The number of insects tested in each concentration group was 100.

[0029] Table 1. Protein concentration settings

[0030] protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Concentration 7 Concentration 8 Cry1Ab 20 40 80 160 200 300 400 500 Vip3Af(mutation) 20 40 80 160 200 300 400 500 Cry1Ab-Vip3Af(mutation) 10 15 25 50 75 100 150 200 Cry1Ab-Vip3Af (unmutated) 20 40 80 160 200 300 400 500

[0031] Negative control group: The preparation method of the negative control is the same as that of Example 1, the only difference being that the plasmid transferred into the E. coli expression strain is a pET32a empty vector without any inserted DNA sequence.

[0032] 3. Determination method: The LC50 of the lethal concentration of the recombinant protein to insects was determined by the surface tiling method.

[0033] After the artificial insect feed is cooked, it is spread flat on a 24-well culture plate (each well has a diameter of 1.5 cm) and the feed is waited for to solidify. In each 24-well plate, 20 wells are randomly set as the experimental group and 4 wells are the negative control group. The surface of the feed in each well of the experimental group is spread with 200 microliters of recombinant proteins of different concentrations in Table 1, and the surface of the feed in each well of the negative control group is spread with 200 microliters of negative control protein. After the entire plate is spread, it is placed on a shaker at 120 rpm and shaken until the liquid dries. Two test insects are placed in each well and sealed with a breathable sealing film. They are kept in a light-shielded incubator at 28°C for 7 days, and the insect mortality rate is counted to obtain the corresponding lethal concentration LC50. The results are shown in Table 2 below.

[0034] Table 2. LC50 of recombinant proteins

[0035]

[0036] Note: 95% CI, 95% confidence interval; N / A, Not active, no biological activity; brackets indicate minimum and maximum values.

[0037] From the above bioassay results, it can be seen that compared with the single Cry1Ab protein and the single Vip3Af mutant protein, the recombinant Cry1Ab-Vip3Af (mutant) has a significantly improved effect and is also highly active against the resistant cowpea pod borer.

[0038] Example 3: Obtaining a plasmid vector containing a fusion protein

[0039] (1) Fusion protein gene expression frame: The insect-resistant fusion protein Cry1Ab-Vip3Af is composed of BT insecticidal crystal protein Cry1Ab and mutated BT vegetative insecticidal protein Vip3Af through fusion connection, and the nucleotide sequence of the coding gene cry1ab-vip3af is SEQ ID NO: 3. The 5' end of the DNA sequence of the fusion protein is connected to the ArUbi promoter (SEQ ID NO: 5), and the 3' end is connected to the Tnos terminator (SEQ ID NO: 6), and the pZmUbi-cry1ab-vip3af-Tnos fragment is artificially synthesized.

[0040] (2) Screening gene expression cassette: The screening gene CP4-EPSPS (SEQ ID NO: 7) was connected to the maize ubiquitin promoter ZmUbi (SEQ NO ID: 8) at the 5' end and the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 9) at the 3' end to artificially synthesize the pZmUbi-CP4-EPSPS-TCaMV35S fragment.

[0041] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ, and a fragment of 6783 bp was recovered. The above two gene expression frames were recombined by seamless cloning and transformed into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector CCV was obtained.

[0042] (4) Agrobacterium transformation: The plasmid CCV is electroporated into Agrobacterium EHA105 competent cells. After enzyme digestion and identification, the cells are preserved in glycerol stock to obtain recombinant Agrobacterium for crop transformation.

[0043] Example 4: Obtaining transgenic cowpea

[0044] The method for obtaining transgenic cowpea is based on the existing technology, using Agrobacterium tumefaciens-mediated transformation method (Chinese patent CN113106118B).

[0045] Scrape the Agrobacterium of Example 3 into B5 liquid culture medium, suspend and mix, use B5 liquid culture medium as blank control, use spectrophotometer (Eppendorf BioPhotometer D30) to detect OD600nm, adjust the concentration of Agrobacterium with B5 liquid culture medium, if the concentration is low, scrape Agrobacterium again until the measured OD600nm value is 0.6, then add final concentration of 2 mg / L 6-BA (6-benzylaminoadenine), 1 mg / L GA3 (gibberellic acid) and 5 mg / L AS (acetosyringone) to obtain Agrobacterium infection solution;

[0046] Sterilized non-transgenic cowpea seeds were placed on MS solid culture medium, germinated and cultured in the dark at 26°C for 2 days, the husks were removed, and the seeds were divided into two parts along the embryonic axis as two explants. 100 explants were immersed in 100 ml of Agrobacterium infection solution and infected at 26°C for 45 minutes;

[0047] The explants infected in the above step were placed on co-culture medium and cultured under constant temperature conditions of 27°C, 5000 Lx, and 16h / 8h light / darkness for 4 days;

[0048] The explants co-cultured in the above step were transferred to a bud induction screening medium, with the hypocotyl end facing downward, and cultured for 28 days under a constant temperature condition of 27°C, 5000Lx, and 16h / 8h light / darkness to obtain explants with new buds;

[0049] The explants with new buds obtained in the above step are transferred to a bud elongation screening medium, and cultured for 28 days under constant temperature conditions of 27°C, 5000Lx, and 16 / 8h light / darkness to elongate positive buds and obtain explants with positive buds;

[0050] The explants with positive buds in the above step were transferred to rooting medium and cultured under constant temperature conditions of 27°C, 5000Lx, and 16 / 8h light / darkness until roots grew out. After culturing for another 5 days, the seedlings were hardened and transplanted to the greenhouse.

[0051] Example 5: Detection of insect-resistant protein in transgenic cowpea transformants and screening of transformants

[0052] Determine the content of insect-resistant protein in the T0 generation cowpea transformed strain obtained by genetic transformation in Example 4. Take 6 strains whose plant phenotypes are not significantly different from those of non-transgenic plants, 50 mg of leaf tissue of each strain at the V6 leaf stage, and take 3 samples of each leaf tissue as replicates; add 500 microliters of PBS buffer after liquid nitrogen grinding, mix thoroughly and centrifuge at 12000 rpm for 5 minutes, dilute the supernatant 500 times and perform enzyme-linked immunosorbent assay (ELISA) determination, the reagent used in the determination is Envirologix's ELISA quantitative kit (product number AP-003), and refer to the product instruction manual for operation.

[0053] The results of the transformation screening of the transformed strains are shown in Table 3 below. The expression data are the average values ​​of three replicates of the strain. Studies in this field have shown that the two proteins in the fusion protein are expressed in equal amounts (Xu et al. 2019). Therefore, the ELISA quantitative kit (product number AP-003) of Envirologix was used to measure the expression of Cry1Ab. The expression of the fusion protein was estimated based on the molecular weight of the protein. The final data are shown in Table 3 below, where CCV1, CCV2, and expression levels are higher.

[0054] Table 3. Protein expression levels of transformed strains

[0055] Transformation strains Cry1Ab determination value ug / g-fwt* Cry1Ab-Vip3Af expression ug / g-fwt* CCV1 9.7 21.5 CCV2 8.6 19.1 CCV3 25.3 55.9 CCV4 41.7 92.3 CCV5 57.8 127.9 CCV6 13.6 30.1 CCV7 7.4 16.4 CCV8 74.1 163.9

[0056] Note: *ug / g-fwt, micrograms per gram of fresh tissue.

[0057] Embodiment 6:

[0058] Take the transgenic cowpea of ​​embodiment 5 to measure the overexpression amount, carry out the indoor biological activity determination of cowpea lepidopteran pests and resistant lepidopteran pests, and therefrom screen out the transformation strain with high insecticidal activity to cowpea pests. Take the fresh plant leaf tissue of 5-6cm length, be placed in a culture dish of 7.5cm in diameter, pad a moist filter paper, access 10 larvae (same as embodiment 2) in the initial hatching 2h, close the culture dish with sealing film after covering the lid to prevent the larvae from escaping. Each transformant is provided with 3 repeated treatment groups, and each bioassay experiment is used as negative control with non-transgenic plants of the same growth period. After 5 days, the survival number of larvae in each experimental group is counted, and the mortality rate of larvae is calculated. According to the mortality data obtained by statistics, the transgenic strain with high resistance to cowpea pests is screened out, and the result is as shown in Table 4.

[0059] Table 4. Pest resistance of transformed cowpea strains

[0060] mortality rate Cowpea pod borer Cowpea pod borer CCV8 100％ 100％ CCV5 100％ 100％ CCV4 100％ 100％ CCV3 100％ 100％ CCV7 75％ 64％ OC1 47％ 0％ CK 0％ 0％

[0061] Note: OC1, cowpea transformant expressing single Cry1Ab protein; CK, control non-transgenic cowpea.

[0062] The results show that the four excellent resistant transformants finally screened out in the present embodiment have high resistance to cowpea lepidopteran pests and plant phenotypes close to non-transgenic ones. The obtained transformants were tested for biological activity against cowpea pod borer and resistant cowpea pod borer, and the insecticidal rate was 100%. Compared with the cowpea transformants expressing a single Cry1Ab protein, the biological activity was significantly improved.

[0063] Finally, the above are only some embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many extensions and expansions. All extensions that can be directly derived or associated from the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A fusion protein for controlling resistant lepidopteran pests of cowpea, characterized in that: The fusion protein is composed of BT insecticidal crystal protein and BT vegetative insecticidal protein through fusion connection, the BT insecticidal crystal protein is BT insecticidal crystal protein Cry1Ab; the BT vegetative insecticidal protein is the mutated BT vegetative insecticidal protein Vip3Af, and the nucleotide sequence is shown in SEQID NO:

2.

2. The fusion protein according to claim 1, characterized in that The BT insecticidal crystal protein Cry1Ab has a nucleotide sequence as shown in SEQ ID NO:

1.

3. The fusion protein according to claim 1, characterized in that The nucleotide sequence of the fusion protein is shown in SEQ ID NO:

3.

4. A recombinant DNA molecule of the fusion protein according to claim 1, characterized in that: The recombinant DNA molecule comprises the fusion protein encoding nucleotide sequence and a promoter sequence for starting the expression of the fusion protein in plants.

5. A DNA construct comprising the recombinant DNA molecule of claim 4.

6. A method for controlling cowpea lepidopteran pests and resistant lepidopteran pests using the fusion protein of claim 1, characterized in that: The method comprises: contacting the fusion protein with the target pest, or transferring the fusion protein into the plant genome, so as to achieve the purpose of preventing and controlling the target pest.

7. The method according to claim 6, characterized in that The target pests include lepidopteran pests or resistant lepidopteran pests.

8. The method according to claim 7, characterized in that The lepidopteran pests include cowpea pod borer, and the resistant lepidopteran pests include resistant cowpea pod borer insensitive to Cry toxins.

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