CN115843833A - Application of insect galectin in lepidoptera pest control - Google Patents

Abstract

The invention belongs to the technical field of agricultural pest control, and particularly relates to application of insect galectin in preventing and controlling lepidoptera pests. In order to research the method for improving the efficiency of the fungal pesticide, the invention discovers that the half-lethal time of the prodenia litura treated by combining the galectin protein and the Nomuraea rileyi is obviously shortened and the pesticidal efficiency is improved compared with the method for treating the prodenia litura by only using the Nomuraea rileyi, and the invention proves that the pesticidal efficiency of the pathogenic fungi (the Nomuraea rileyi) can be obviously improved and the pesticidal effect of the fungal pesticide can be accelerated by matching the galectin protein and the Nomuraea rileyi.

Description

Application of insect galectin in preventing and controlling lepidoptera pests

Technical Field

The invention belongs to the technical field of agricultural pest control, and particularly relates to application of insect galectin in preventing and controlling lepidoptera pests.

Background

Galectin is a protein containing a binding site for beta-galactoside (β -galactoside), is widely present in animals, and is involved in various physiological functions such as inflammation, immune response, cell migration, autophagy, and signal transduction. In mammals, various diseases are associated with galectins, such as fibrotic lesions, cancer, heart disease, and the like. However, few studies have been made on the galectins of insects, and some studies have shown that the galectins of insects may participate in the recognition of bacteria. However, there is no study on the function of galectins in entomopathogenic fungal infections.

Spodoptera litura Fabricius is a crop pest of Lepidoptera Noctuidae, and takes 300-plant leaves of sweet potatoes, cotton, soybeans and the like, which are rampant intermittently. At present, the control of prodenia litura mainly depends on chemical pesticides, but the problems of pest drug resistance, pesticide residue, environmental pollution and the like are caused by the large-scale use of the chemical pesticides, so that the healthy life of human beings and the sustainable development of agriculture are seriously influenced. Nomuraea rileyi (Nomuraea rileyi) is a pathogenic fungus of insects, frequently causes epidemic diseases of various pests such as prodenia litura, cotton bollworm, soybean looper and the like in fields, and particularly has stronger pathogenicity to noctuidae pests. Although the fungal pesticide is environment-friendly and is not easy to generate drug resistance, the pesticidal effect is slow, so that the popularization and the application of the fungal pesticide are restricted. Therefore, the method for improving the efficiency of the fungal pesticide has important economic value.

Disclosure of Invention

In order to overcome the defects of the prior art, the galectin protein and the Nomuraea rileyi are combined, so that the insecticidal toxicity of the Nomuraea rileyi is enhanced, and the insecticidal effect of the Nomuraea rileyi is accelerated.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of galectin protein in preventing and treating lepidoptera pests.

The invention also provides application of the galectin protein in preparing a pesticide for preventing and treating lepidoptera pests.

Preferably, the lepidopteran pest is a prodenia litura.

Preferably, the galectin protein is prodenia litura galectin-12 (SlGale 12) protein, and the accession number of the SlGale12 protein NCBI is LOC111359284 and has an amino acid sequence shown in SEQ ID No. 6.

According to the invention, researches show that after Nomuraea rileyi infects spodoptera litura larvae, the expression level of SlGale12 genes in larva haemolymph is obviously increased, and after the Spodoptera litura is treated by a mixture of SlGale12 protein and Nomuraea rileyi, the semi-lethal time LT50 of the Spodoptera litura can be shortened from 121.636 +/-1.589 hours to 113.600 +/-1.391 hours, so that the insecticidal effect is obviously improved. The SlGale12 protein can enhance the insecticidal toxicity of Nomuraea rileyi to prodenia litura and accelerate the insecticidal effect of the Nomuraea rileyi.

More preferably, the biological material related to the SlGale12 protein is also included, the biological material includes a gene encoding the SlGale12 protein, an expression cassette of the SlGale12 gene, a recombinant vector of the SlGale12 gene, a recombinant microorganism of the SlGale12 gene, and a recombinant cell line of the SlGale12 gene, and the SlGale12 gene has a nucleotide sequence shown as SEQ ID No. 5.

The invention also provides a pesticide for preventing and treating prodenia litura, which comprises galectin protein and Nomuraea rileyi.

Preferably, the galectin protein is SlGale12 protein, and the SlGale12 protein has an amino acid sequence shown in SEQ ID NO. 6.

Preferably, the Nomuraea rileyi is a spore suspension of Nomuraea rileyi.

The invention also provides a method for preventing and controlling prodenia litura, which is to accelerate the insecticidal efficiency of the prodenia litura by using the galectin protein and the Nomuraea rileyi in a combined manner.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses application of galectin in preventing and controlling lepidoptera pests, and finds that the half-lethal time of combined treatment of spodoptera litura is obviously shortened and the insecticidal efficiency is improved by treating the spodoptera litura larvae with a mixture of galectin protein and Nomuraea rileyi compared with the method of treating the spodoptera litura larvae with the Nomuraea rileyi only.

Drawings

FIG. 1 shows the expression level changes of SlGale12 after Nomuraea rileyi infection;

FIG. 2 shows the cDNA cloning and vector construction verification results of Spodoptera litura SlGale12 (A is the cDNA amplification result of SlGale 12; B is the plasmid restriction enzyme digestion verification result of pET28a-SlGale 12);

FIG. 3 is an SDS-PAGE pattern of prokaryotic expression SlGale12 protein;

FIG. 4 shows the effect of SlGale12 protein addition on the pesticidal rate of Nomuraea rileyi.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 Change in expression level of Trichoplusia littoralis SlGale12 Gene after infection with Nomuraea rileyi

(1) Experimental methods

The prodenia litura larvae are cultured in an artificial climate incubator (the temperature is 27 +/-2 ℃, the relative humidity is 50 +/-10%, the photoperiod is day: night = 13h). Nomuraea rileyi Nr (purchased from Mingrophyte) was inoculated into SMAY medium, grown at 25 ℃ for 10 days, and conidia of Nomuraea rileyi were collected with 0.05% Tween20 solution. Then Nomuraea rileyi (5X 10) ⁵ Spores/worms) were injected into 5 th day spodoptera litura larvae and injected at 0.05% Tween20 as control, hemolymph from control and treatment groups were collected after 24, 48 and 72 hours, with 3 replicates per group, each replicate consisting of 3 larvae. Extracting total RNA, carrying out reverse transcription to obtain cDNA, and finally adopting primers Gale12-qF (CACACGCCCTATTCCTGGAG, SEQ ID NO. 1) and Gale12-qR (GAAGCGAGGGTTGAAGTGGA, SEQ ID NO. 2) to carry out quantitative PCR analysis on the expression quantity of the gene. For specific operation, reference is made to the literature "Lu Y, su F, zhu K, et al, synthetic genetic analysis of C-type light-domain genes in seven holobolous infections [ J].Insect Biochemistry and Molecular Biology,2020,126:103451”。

(2) Results of the experiment

As shown in the results of fig. 1, compared to the control group, the expression level of the prodenia litura Gale12 gene was significantly increased after 24 hours of noctuid infection, the expression level of SlGale12 continued to increase after 48 hours, and the expression level of SlGale12 decreased after 72 hours, but still significantly higher than that of the control group.

Example 2 construction of Prodenia litura SlGale12 expression vector

(1) Experimental methods

And taking cDNA of prodenia litura blood cells as a template, and carrying out PCR amplification on the SlGale12 fragment by adopting Gale12-F (CGGATCCATGACTACAATTGTAAATCC, SEQ ID NO. 3) with BamHI and Gale12-R (GTCTCGAGGGCGTCCGCGAAGGTTACGT, SEQ ID NO. 4) primers with XhoI. The PCR system is as follows:

max DNA Polymerase 25. Mu.L, gale12-F (10. Mu.M) 1. Mu.L, gale12-R (10. Mu.M) 1. Mu.L, cDNA template 3. Mu.L, ddH ₂ O20. Mu.L. The amplification procedure was: pre-denaturation at 98 ℃ for 1min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; further extension was carried out at 72 ℃ for 10min. The PCR product was run through agarose gel electrophoresis, the band of interest was excised in a gel cutter, and gel recovery was performed using the gel recovery kit from OMEGA according to the instructions.

The pET28a (Saimerfin) and SlGale12 fragments were each double digested with Fast digest BamHI and XhoI in a water bath at 37 ℃ for 1 hour. And recovering and purifying the SlGale12 enzyme-digested fragment by utilizing an OMEGA Cycle-Pure kit according to the instruction, cutting a target strip on a gel cutting instrument after the plasmid pET28a enzyme-digested fragment is subjected to agarose gel electrophoresis, and recovering gel by utilizing an OMEGA gel recovery kit according to the instruction. Then, T4 ligase is used for connecting the plasmid and the fragment after enzyme digestion at 16 ℃ overnight, the connection product is transformed into a Top10 competent cell, shake culture is carried out after positive single bacteria are screened by PCR, and the plasmid is sent to the department of biology company for sequencing verification after being extracted. The plasmid with the correct sequencing is transformed into BL21 competent cells, and a single colony (pET 28a-SlGale12 expression strain) is selected and stored in glycerol and is reserved at-80 ℃.

(2) Results of the experiment

As shown in FIG. 2A, a cDNA fragment of SlGale12 gene was obtained by PCR amplification, and the size was about 430bp. As shown in FIG. 2B, digestion of pET28a-SlGale12 plasmid verified that a SlGale12 fragment of approximately 430bp in size could be excised. The nucleotide sequence of the SlGale12 obtained by sequencing is shown in SEQ ID NO.5, and the sequence of the predicted SlGale12 protein is shown in SEQ ID NO. 6.

SlGale12 cDNA(SEQ ID NO.5)：

ATGACTACAATTGTAAATCCGCAAGTACCATTCACACGCCCTATTCCTGGAGGTTTATCCCCCGGCCGCACTATAACTATCGAGGGTGCCATCCCTCCCCGATCAGACAGGTTCGCAATAAACTTGCAATGTGGTAGCGCGGACATCGCTTTCCACTTCAACCCTCGCTTCGGAGATCAGTGCATAGTTCGCAACTCCTATATTTCTGGCCACTGGGGTGCTGAGGAGACCAGTGGAGGCATGCCGTTGGTTAGGGGAGAACAGTTTGAAGCTGAATTTAAGTGCTCTGAAGGTCATTTTACGGTGAATTTGAACGGGAAACATTTTTGCGACTTCCCACACCGCATCCCATACCATAAGATCAACCACATCAACGTGGACGGTGATGTCAACATAAGGCACGTAACCTTCGCGGACGCCTGA。

SlGale12 protein (SEQ ID NO. 6):

MTTIVNPQVPFTRPIPGGLSPGRTITIEGAIPPRSDRFAINLQCGSADIAFHFNPRFGDQCIVRNSYISGHWGAEETSGGMPLVRGEQFEAEFKCSEGHFTVNLNGKHFCDFPHRIPYHKINHINVDGDVNIRHVTFADA。

example 3 prokaryotic expression and purification of Spodoptera litura SlGale12 protein

(1) Experimental methods

The pET28a-GFP expressing strain (Wu J, lin J F, wang X C, et al, pressure-depleted magnetic and structural transitions of the component of iron-based 122superconductors BaFe2As2J., [ 2 ] is used as a vector for expressing the protein].Proc Natl Acad Sci U S A,2013,110(43) 17263-17266.) As a control, pET28a-GFP and pET28a-SlGale12 expression strains were inoculated in LB liquid medium containing 50. Mu.g/mL kanamycin, respectively, and shake-cultured at 220rpm on a shaker at 37 ℃ until OD ₆₀₀ Is 0.5. Then, IPTG was added to the medium to a final concentration of 0.1mM, and the culture was continued at 37 ℃ and 220rpm for 4 hours. Centrifuging at 5000rpm for 5min at room temperature, and removing supernatant to obtain thallus. 20mL of 1 XPBS was added to resuspend the cells, the cells were disrupted on ice using an ultrasonicator, and the mixture was centrifuged at 8000rpm for 15min at 4 ℃ to obtain a supernatant.

The protein in the supernatant was purified using Ni NTA Beads (Tiandi human and Bio Inc.) according to the instructions and eluted with 500mM imidazole to give a protein solution. Then SlGale12 and GFP eluates were dialyzed against 1 XPBS for 3 times, and the resulting protein solutions were sterilized by filtration (0.5 mg/mL) and stored at-80 ℃ until use.

(2) Results of the experiment

The resulting pET28a-GFP and pET28a-SlGale12 proteins were analyzed by SDS-PAGE. As shown in FIG. 3, the pET28a-GFP and pET28a-SlGale12 proteins obtained by purification were approximately 30kDa and 20kDa, respectively, and corresponded to the expected sizes.

Example 4 Effect of SlGale12 protein on the insecticidal efficiency of Nomuraea rileyi

(1) Experimental methods

Nomuraea rileyi Nr-06 was inoculated into SMAY medium, grown at 25 ℃ for 10 days, and conidia of Nomuraea rileyi were collected with 0.05% Tween20 solution. The prodenia litura larvae L5D1 of the first day of five ages are divided into 4 groups, 50 pieces of each group are injected with SlGale12 protein, GFP protein, mixture of SlGale12 and Nomuraea rileyi, and mixture of GFP protein and Nomuraea rileyi respectively. The protein dosage in the above treatment is 3 μ g/insect, and the Nomuraea rileyi dosage is 5 × 10 ⁴ Spores/insect. The death number of the spodoptera litura larvae is recorded every day, and after all larvae in the experimental group are dead, a survival curve is drawn.

(2) Results of the experiment

As shown by the results in fig. 4, treatment of larvae with SlGale12 or GFP protein alone did not affect survival of larvae. After the larvae are treated by the mixture of GFP and Nomuraea rileyi, the half-lethal time LT50 is 121.636 +/-1.589 hours, and after the prodenia litura is treated by the mixture of SlGale12 protein and Nomuraea rileyi, the half-lethal time LT50 is 113.600 +/-1.391 hours, which shows that the SlGlae12 protein promotes the infection speed of the Nomuraea rileyi on the larvae and accelerates the death of the larvae.

In conclusion, the SlGale12 protein and the Nomuraea rileyi are combined, so that the insecticidal toxicity of the Nomuraea rileyi can be effectively enhanced, and the insecticidal effect is remarkably accelerated.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (9)

1. Application of galectin protein in preventing and treating lepidoptera pests.
2. Application of galectin protein in preparing insecticide for preventing and treating lepidoptera pests.
3. 3. Use according to claim 1 or 2, wherein the lepidopteran pest is a prodenia litura.
4. 4. The use according to claim 1 or 2, wherein the galectin protein is SlGale12 protein, and the SlGale12 protein has an amino acid sequence shown as SEQ ID No. 6.
5. 5. The use according to claim 4, further comprising biological material related to the SlGale12 protein, wherein the biological material comprises a gene encoding the SlGale12 protein, an expression cassette of the SlGale12 gene, a recombinant vector of the SlGale12 gene, a recombinant microorganism of the SlGale12 gene, and a recombinant cell line of the SlGale12 gene, and the SlGale12 gene has a nucleotide sequence shown as SEQ ID No. 5.
6. 6. An insecticide for controlling prodenia litura, which comprises galectin protein and Nomuraea rileyi.
7. 7. The pesticide of claim 6, wherein the galectin protein is SlGale12 protein, and the SlGale12 protein has an amino acid sequence shown as SEQ ID No. 6.
8. 8. The pesticide for controlling prodenia litura according to claim 6, wherein the Nomuraea rileyi is a spore suspension of Nomuraea rileyi.
9. 9. A method for controlling prodenia litura is characterized in that the insecticidal efficiency of prodenia litura by Nomuraea rileyi is accelerated by combining galectin protein and Nomuraea rileyi.

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