A harpocera lesueuri cocoon bee venom protein hhmapk15 and application thereof

Abstract

The present application relates to a kind of HhMAPK15 of anthocarposa larva parasitoid venom protein and its application, it relates to the field of protein engineering, its amino acid sequence is as shown in SEQ ID NO:1, the protein can be used to inhibit pest larva to continue development.The present application will be used to inhibit pest larva to continue development on anthocarposa larva parasitoid venom protein HhMAPK15, can reduce the disease of crop by pest to a certain extent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, and in particular to a venom protein HhMAPK15 of Microplitis tolteca and application thereof. BACKGROUND

[0002] "Food in hand, heart not panic", agriculture can not be ignored and weakened at any time. "The country is based on agriculture, the people are based on food, and the food is based on safety first". Crop pest control is closely related to agricultural food, and crop production history is accompanied by natural disasters. As early as 700 BC, there were records of insect control in China. There are more than 1600 harmful organisms that harm crops in China, of which there are 830 species of pests. With the rapid development of modern agriculture, the degree of agricultural production intensification is becoming higher and higher, and the trend of increasing crop production and increasing resistance of pests to insecticides and other chemical agents is increasing year by year, prompting us to find green and efficient pest control technology.

[0003] Natural enemy insects and their genetic resources have important application prospects in pest control. Parasitic wasps can be directly used for field or storage pest control. However, the high adaptability of parasitic wasps to hosts and the influence of changes in field temperature and humidity on the survival of parasitic wasps will greatly limit their control effect and range of action. The development and utilization of insecticidal proteins in parasitic wasp venom can avoid the influence of parasitic behavior, and can affect a variety of pests. For example, the venom of Microplitis tolteca can paralyze the larvae of many pests of the family Pyralidae and Noctuidae, and is not affected by the selection of its host. At the same time, the venom of parasitic wasps is different from that of toxic species such as spiders, and generally has no effect on the nervous system of mammals, and is safer for humans and animals. In addition, venom proteins can be used to create insect-resistant genetically engineered crops or improve insecticidal microorganisms; after the combination of venom protein macromolecules and nanomaterials, the host pests can be killed by feeding.

[0004] The present application relates to the technical field of protein engineering, and in particular to a venom protein HhMAPK15 of Microplitis tolteca and application thereof. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application aims to provide a venom protein HhMAPK15 of Microplitis tolteca and application thereof, which can inhibit the development of larvae of Lepidoptera Noctuidae and Pyralidae pests.

[0006] The above application object of the present application is achieved by the following technical scheme:

[0007] A venom protein HhMAPK15 of Microplitis tolteca, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] The venom protein HhMAPK15 of the said Achroia grisella has the use of inhibiting the further development of pest larvae.

[0009] The said pest larvae are the larvae of the Lepidoptera Pyralidae Indian meal moth.

[0010] The present application uses the venom of the Achroia grisella parasitizing artificial host to obtain the venom injected into the host by the parasitoid, through proteomic detection, the protein mitogen-activated protein kinase HhMAPK15 is identified in the venom component, the full-length sequence and amino acid sequence of the protein mitogen-activated protein kinase HhMAPK15 gene are obtained by combining the monohedric genome of the Achroia grisella and the venom gland transcriptome, after the prokaryotic expression and the purification under the non-denaturing condition, the fusion protein with the TF solubilization tag is obtained, and the purified HhMAPK15 is obtained by removing the solubilization tag through thrombin, the direct injection of the HhMAPK15 into the Indian meal moth larvae can inhibit the development rate of the Indian meal moth, prolong the time of developing into pupae, not only make the development of the Indian meal moth abnormal, but also affect the eclosion of the Indian meal moth. The venom protein HhMAPK15 of the Achroia grisella is used for inhibiting the further development of pest larvae, which can reduce the damage of the pests to crops to a certain extent.

[0011] In summary, the present application has at least one of the following beneficial technical effects:

[0012] 1. The venom protein HhMAPK15 of the Achroia grisella is used for inhibiting the further development of pest larvae, which can reduce the damage of the pests to crops to a certain extent;

[0013] 2. In particular, the development rate of the Indian meal moth can be inhibited, and the time of developing into pupae can be prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the expression and purification of the recombinant protein, (A) the SDS-Page diagram and Western blot diagram of the purified recombinant protein His-TF-HhMAPK15, and (B) the SDS-Page diagram and Western blot diagram of the recombinant protein His-TF-HhMAPK15 after enzyme cutting, wherein the red triangle represents the recombinant protein HhMAPK15-His;

[0015] Figure 2 is the time of developing into pupae of the Indian meal moth larvae after injecting the prokaryotic expression recombinant protein His-HhMAPK15 and His-GFP, (A) the record of the time of developing into pupae of the Indian meal moth larvae, and (B) the photos of the Indian meal moths in 0-4d;

[0016] Figure 3 Figure 3 is a chart showing the changes in the developmental hormone titers of the host after injection of the prokaryotic expression fusion protein HhMAPK15-His and His-GFP, wherein the difference comparison analysis is as follows: ns, no significant difference (p>0.05); *, significant difference (p<0.05); **, extremely significant difference (p<0.01); ***, extremely significant difference (p<0.001). DETAILED DESCRIPTION

[0017] The application will be further described in detail below with reference to the accompanying drawings.

[0018] EMBODIMENT

[0019] Expression, purification and antibody preparation of recombinant protein

[0020] 1. Prokaryotic expression using the pCold TF system:

[0021] 1) Construction of recombinant plasmid

[0022] The HhMAPK15 nucleic acid fragment obtained by PCR (in combination with the Pfam database and the early research results of the inventors, HhMAPK15 is derived from the genome of Cotesia rubecula, and the nucleic acid sequence is shown as SEQ ID NO: 2);

[0023] The linear pCold TF vector is cut using the endonuclease Eco53kI (NEB, USA) and HindIII enzyme (NEB, USA);

[0024] The HhMAPK15 fragment is inserted into the plasmid by homologous recombination using the plus One step PCR Cloning Kit (Novoprotein Scientific Inc., Suzhou, China);

[0025] The primers are shown in Table 1 of Appendix I.

[0026] Table 1 of HhMAPK15 and GFP recombinant plasmid primers

[0027]

[0028] 2) Transformation of recombinant plasmid

[0029] The constructed pColdTF_HhMAPK15 plasmid is transformed into DH5a competent cells (GenScript, Nanjing, China), and the correct positive clone is obtained through ampicillin screening, colony PCR and sequencing verification, and the plasmid is extracted;

[0030] The plasmid was transformed into BL21 competent cells (GenScript, Nanjing, China), and positive clones were screened and stored.

[0031] 3) Inducing expression of recombinant protein

[0032] The bacterial solution was expanded in a 37°C incubator at 200 rpm until the OD 600 of the bacterial solution reached about 0.6, and isopropyl-β-D-thiogalactoside (IPTG) (Sangon Biotech, Shanghai, China) was added to a final concentration of 1 mM. The bacterial solution was transferred to a 16°C, 120 rpm incubator for 16 hours of expression induction.

[0033] The bacterial solution was then centrifuged (8000g, 5 min), and the precipitate was washed with PBS three times to remove as much culture solution as possible. The bacterial solution was stored at -80°C.

[0034] 4) Lysis of bacterial solution

[0035] The bacterial solution was resuspended with a 1:5 ratio of buster Master Mix at room temperature, and the bacteria were fully lysed by shaking for 20 min.

[0036] 5) Purification of protein and preparation of antibody

[0037] His-Bind Purification Kit (Novagen) and Tris-HCl buffer (pH 7.44) were used for purification. After desalting, His-TF-HhMAPK15 was cleaved with thrombin (Yuanye Bio-Technology Co., Ltd, Shanghai, China) to remove the solubilizing tag His-TF, and HhMAPK15-His was obtained and purified again. The protein concentration was determined, and the protein was stored at -80°C.

[0038] HhMAPK15 rat antibody was prepared by Huaan Biotechnology.

[0039] 2) Expression of recombinant protein and enzyme cleavage

[0040] His-TF-HhMAPK15 protein was expressed using E. coli, and the entire recombinant protein was about 126.40 kDa Figure 1 (A). Western blotting confirmed that the protein molecular size was as expected. In this study, thrombin was used to remove the solubilizing tag (His-TF), and HhMAPK15-His was purified. The results of protein gel electrophoresis and Western blotting are shown in Figure 1 (B). HhMAPK15 was about 73.06 kDa, which was basically consistent with the predicted protein molecular weight. The HhMAPK15 amino acid sequence is shown in SEQ ID NO: 1.

[0041] 3 Recombinant protein injection of host

[0042] 1 μg / μL of His-GFP and HhMAPK15-His proteins were injected into 5th instar late Chorotozona roboralis using a microinjector (Nanoject II, Drummond scientific company broomall PA, USA) with a volume of 1 μL per head.

[0043] 4 Function of recombinant protein

[0044] 1 μg of recombinant protein (HhMAPK15-His and His-GFP) was injected into 5th instar Chorotozona roboralis larvae, and the time of pupation of Chorotozona roboralis larvae was observed and photographed. Figure 2 ) It was found that the larvae developed pupae successively after 2.5 d, and 16.67±2.89% of the pupae of Chorotozona roboralis larvae injected with recombinant protein HhMAPK15-His (treated group larvae) were imperfect. In addition, Chorotozona roboralis larvae injected with recombinant protein His-GFP (control group larvae) developed pupae faster than treated group larvae (p=0.0039) Figure 2 ). However, from the detection of juvenile hormone and ecdysone titers, the ecdysone titer of the treated group larvae was significantly higher than that of the control group larvae at 12 h, and there was no significant difference in the later stage; while the titers of the three juvenile hormones in the treated group larvae were generally higher than those in the control group larvae Figure 3 ). This may be the reason for the slow development of the treated group larvae.

[0045] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, and the modifications are protected by the patent law as long as they are within the scope of the present application.

Claims

1. A venom protein of H. hebetor, HhMAPK15, characterized in that, The amino acid sequence thereof is shown as SEQ ID NO:

1.

2. Use of the venom protein HhMAPK15 of Microplitis tolteca for inhibiting the pupation of Chilo suppressalis (Lepidoptera: Pyralidae) larvae.

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