CN116813739A - Spodoptera litura lethal gene tin and application thereof - Google Patents
Spodoptera litura lethal gene tin and application thereof Download PDFInfo
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- CN116813739A CN116813739A CN202310911843.6A CN202310911843A CN116813739A CN 116813739 A CN116813739 A CN 116813739A CN 202310911843 A CN202310911843 A CN 202310911843A CN 116813739 A CN116813739 A CN 116813739A
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- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a prodenia litura lethal gene tin and application thereof. The invention aims to provide a new choice for biological insect control. The technical scheme of the invention is that the coded protein of the prodenia litura lethal gene tin has an amino acid sequence shown as SEQ ID No. 2. The invention also provides application of the prodenia litura lethal gene tin or/and the coding protein in insect control. According to the invention, by reducing the expression of the gene tin in the prodenia litura larvae, the larva development is inhibited, even death is caused; provides a new choice for realizing green and accurate prevention and control of prodenia litura.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a prodenia litura lethal gene tin and application thereof.
Background
Prodenia litura Spodoptera litura (Fabriius) belongs to the Lepidoptera noctuidae, is an important agricultural pest distributed worldwide, can seriously harm various important cash crops such as cotton, soybean, tobacco and the like, and causes serious loss to the cash crops in Asia, africa, south America and some oceangoing regions. Prodenia litura mainly occurs seriously in southeast areas of China. Prodenia litura is a main pest in the middle and later stages of tobacco growth, and huge losses can be caused to tobacco farmers when serious pests occur. In order to reduce economic loss, prodenia litura is usually controlled by chemical control in production, but long-term use of one or a class of chemical agents often leads to continuous enhancement of drug resistance of the prodenia litura, and the risk of pesticide residue exists in blade maturity. Therefore, there is an urgent need in production to create new environment-friendly methods for controlling prodenia litura.
RNA interference (RNAinterference, RNAi) refers to the highly conserved, double-stranded RNA (double-strandedRNA, dsRNA) -induced, highly efficient, specific degradation of homologous mRNA during evolution. The pest control technology based on RNAi can prevent the normal growth, development and reproduction of pests and even lead to the death of the pests directly by interfering with the key genes for controlling the development or important behaviors of the pests, thereby achieving the purpose of pest control. RNA-based pest management strategies are now being used for the control of some insects and viruses, and possibly for the control of other pests such as weeds, nematodes, fungi and bacteria in the future. RNAi has the potential to effectively control pesticide resistance in agricultural pests and disease vectors, and to destroy animal and plant viral vectors, as well as control invasive species. The dsRNA is ubiquitous in organisms and is easy to degrade in the environment, so the dsRNA is nontoxic, has no residue and no pollution to the environment, is a novel environment-friendly pest control method, and has very broad application prospect.
Carbon quantum dots (Carbon quantum dots, CQDs) are nanoparticles having a particle diameter of 10nm or less, and have been attracting attention in recent years because of their excellent water solubility and extremely low toxicity.
Disclosure of Invention
The invention aims to provide a new choice for biological insect control.
The technical scheme of the invention is that the coded protein of the prodenia litura lethal gene tin has an amino acid sequence shown as SEQ ID No. 2.
Furthermore, the invention also provides a prodenia litura lethal gene tin, the nucleotide sequence of which is shown as SEQ ID No.1 or SEQ ID No. 12.
The invention also provides application of the prodenia litura lethal gene tin or/and the coding protein in insect control.
Further, the insect is an insect of the genus noctuid.
Further, the insect is prodenia litura.
The invention also provides a method for preventing and controlling insects, which mainly comprises the step of reducing the expression of gene tin in the insects.
Furthermore, RNAi technology is adopted to reduce expression of gene tin in insects.
In particular, the nucleotide sequence of the dstin is shown as SEQ ID No. 11.
Specifically, the primer sequences for amplifying the dstin are shown as SEQ ID No.3 and SEQ ID No. 4.
Specifically, dsetin is used for injection and feeding of insects.
Wherein the injection is of 2-year-old larvae.
Further, dsRNA was mixed with carbon quantum dots and injected.
Specifically, the concentration of the dsRNA and the concentration of the carbon quantum dots are 100 ng/. Mu.L.
Preferably, the amount of carbon quantum dots is overloaded.
Specifically, the feeding is feeding of a bacterial liquid expressing dstin.
In particular, the bacterial liquid is obtained as follows: the dstin shown in SEQ ID No.11 is constructed on an expression vector, then escherichia coli is transformed, bacterial cells are inactivated after culture, and the precipitate is collected by freeze centrifugation.
Further, the expression vector is an L4440 vector.
Further, the insect is an insect of the genus noctuid.
Further, the insect is prodenia litura.
The invention also provides a substance for reducing gene tin expression, which is dsRNA for interfering gene expression by RNAi.
Specifically, the primer sequences for amplifying the dstin are shown as SEQ ID No.3 and SEQ ID No. 4.
In particular, the nucleotide sequence of the dstin is shown as SEQ ID No. 11.
The invention also provides application of the substance for reducing gene tin expression in insect control.
The invention has the beneficial effects that: the dsRNA for the tin gene synthesized by the invention can inhibit larva development and even die after the second-instar larva of the prodenia litura is injected. RT-qPCR verifies that the expression level of the tin is down-regulated after 48 hours of injection of the second-instar larvae. And the subsequent method of adding carbon quantum dots into the dsRNA can prevent the dsRNA from being degraded and improve the interference efficiency. The dsRNA is designed aiming at specific genes of the prodenia litura, does not change the genome of the prodenia litura, only has the effect of killing the prodenia litura, has the advantages of strong specificity, high safety, high mortality, environmental friendliness and the like, can realize green and accurate prevention and control of the prodenia litura, and can effectively reduce the damage of chemical pesticides to ecological environment. The invention also introduces the nano material (carbon quantum dot) as the carrier of the dsRNA, thereby effectively improving the silencing efficiency of the dsRNA.
Drawings
FIG. 1 effect of RNA interference on Spodoptera litura mortality; dstin represents dsRNA designed against the gene tin; dsGFP represents dsRNA designed for the gene GFP.
FIG. 2 is a graph showing the silencing effect of dsRNA on spodoptera litura ATPd gene after 48h injection; the ordinate indicates the relative expression level of the gene.
FIG. 3 is a schematic diagram of a carbon quantum dot structure.
FIG. 4 electrophoresis detection results after Carbon Quantum Dots (CQDs) are combined with dsRNA; lanes 1 are control bands, 6 bands are 100bp, 250bp, 500bp, 750bp, 1000bp and 2000bp respectively; the other lanes are electrophoresis bands after CQDs and dsRNA are uniformly mixed according to the volume ratio of dsRNA to CQDs of 1:0, 2:1, 3:1, 4:1, 5:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 and 20:1.
FIG. 5 silencing effect of dsRNA on prodenia litura tin gene after 48h of nano-carrier mediated injection; the ordinate indicates the relative expression level of the gene.
FIG. 6L 4440 vector constructed from tin.
Fig. 7, titin bacteria liquid fed prodenia litura mortality.
Detailed Description
In the early stage of the experiment, searching all studies related to insect RNAi through a Web of Science database, searching documents, screening genes with the lethal effect higher than 50% on target pests (such as lepidoptera, diptera, hymenoptera and the like) of various purposes in articles, obtaining 66 genes with the mortality rate higher than 50%, searching homologous genes of related genes in prodenia litura through NCBI, and finding genes which are highly homologous to the prodenia litura from a series of homologous genes as candidate target genes. Still further, RNA-Seq high throughput sequencing technology is adopted to analyze the genes differentially expressed by the prodenia litura from the egg stage to the adult stage, and the genes up-regulated in 1-age, 2-age and 3-age are screened from the up-regulated genes. Finally, 40 genes of prodenia litura are obtained through co-screening to serve as candidate genes.
Then, dsRNA is designed for 40 candidate genes respectively, and the two-instar larvae of the prodenia litura are injected by a microinjection method, and 100nL of each larva is injected, so that the lethality of the tin gene can reach a higher level, and other genes cannot produce a lethality effect on the prodenia litura.
the tin gene is an actin gene, and the protein produced by translation is a protein with molecular weight exceeding 3MDa, is the third most abundant protein in muscle (next to actin and myosin), is responsible for most of the elasticity of myofibrils in various organisms, but has no research on the function of the lepidopteran tin gene.
TABLE 1 primers used in the examples below
EXAMPLE 1RNA interference sequence Synthesis of Titin
Designing dsRNA Primer dstin F/dstin R in the CDS region of the tin gene by utilizing Primer5 according to the sequence (accession number LOC 111364780) of the tin gene queried on the NCBI website; a T7 promoter sequence was added to the 5' end of the primer sequence.
SEQ ID No.1 tin sequence
TTTCACACAACCAGTCTTCGGCGAACGAACGAAGACACACAGCAACATGAAGTTTTTGGTTGTATTGGTCGCTTCGCTGGCCGTGGTGGCCTTTGCCAGCGAGGAGAAAGGCGCACCGAAACAAGCGGCAGCATCCGAAGATAAGAAGCAGGAGAAGAGAGGAATTTATGACTTTGGTCATTATGGAGGCGGTGGTGGCAGCAGCGAGGGAGGTTATGGAGGACATGGTTTCGGCGGTGGCGAAAGTGTGAGCTTCGGACACGGCGGTGGTGAAAGCTTCGGTCATGGAGGTGGCGAGAGCTTCGGACACGGCGGCGGTGACAGCTACGGACAAGGCGGTGGTGACAGCTACGGACATGGTGGCGGTGACAGCTACGGACATGGTGGCGGTGACTTCGGTGGCTCCAGCGGTGGCTGGAAACCCATTGCCTCTGATGAAGGCCACGGACACCATCACCACCAACACCACGAACACGTCAAGACCATCGAAGTCATCAAGAAGGTCCCTGTCCCATACACCGTTGAGAAGCACGTCCCCTACACCGTTGAGAAGAAAGTACCTTATGAAGTCAAAGTACACGTGCCCCAGCCCTACACCATCGAAAAGAAAGTCCCCGTCACCGTGAAGGAGTACGTCAAATACCCCGTGTACGTGCCTGAGCCCTACACCGTTGAGAAGAAGGTACCATATGAAGTCAAGGTCCCCGTCGACAAGCCCTACGAAGTGAAGGTTAAGGTGCCCACTCCTTACACCGTCGAAAAGAAGGTCCCCTATGAAGTCAAGGTCCCCGTACCCCAGCCCTACACTGTCGAGAAGAAGGTCCCAGTCCCCGTGAAATACGAAGTCAAAGTACCCCAACCCTACGAAGTCGTCAAGAAGGTGCCTTATGAAGTTAAAGTGCCCGTCGACAAGCCCTACAACGTGTACGTGCCCAAACCTTACCCAGTCCCTGTTGAGAAGCCCTACCCCGTCACAGTCCACAAGCCTGTGCCCTATGAAGTCAAAGTCCCCGTTGACAAGCCTTACAAGGTTGAGGTTGAGAAGCCCTACCCAGTCCACATCAAGGTCCCAGTGCCCAAACCTTACGACGTGTACAAGAAGATCCCATACACCGTTGAGAAGAAGGTGCCTTTCGAAGTGAAGGTCCCCATTGACAAGCCTTACCCAGTGTACAAGGACGTGCCATTCCCATTGGTGAACCACCACCACGAGCACCACCAGCCCCAGCAACACCACGGTTGGCACTGAGTGACCTTAGGCTAAGTCTCACCTTGTACCTTGTAGGTTAAGTTCAGAAATGTGATCAATTAGTATTTTTAGTGTATTGTATATTTTTGCTAAATTTTGTGAATGTTGGTGCTGTCCGCTCACAGGCCAGTGATGTGACGGCTATGTTGCGGTGAATTGCTGAACGAATTTTGAGTTCTAACTTAAATAGTAAATATGCAAATAAAAATATTCGTAGTTTTGCAGTCGACGAACAATAGGTTGGCCGCATAGCGTCACTTTTGACGCGCGGCCTGCAGTTGTAGATAATTACCTGTTATTATTTTTTTAGCTTTTATATGCTTTGTTATAATGTAACTTGACAAAGTACCTGTACTTTGAGTTGAGGCTGATTTAATAAAAATGTGTGGAATGTTCA
CDS region sequence of SEQ ID No.12 tin gene
ATGAAGTTTTTGGTTGTATTGGTCGCTTCGCTGGCCGTGGTGGCCTTTGCCAGCGAGGAGAAAGGCGCACCGAAACAAGCGGCAGCATCCGAAGATAAGAAGCAGGAGAAGAGAGGAATTTATGACTTTGGTCATTATGGAGGCGGTGGTGGCAGCAGCGAGGGAGGTTATGGAGGACATGGTTTCGGCGGTGGCGAAAGTGTGAGCTTCGGACACGGCGGTGGTGAAAGCTTCGGTCATGGAGGTGGCGAGAGCTTCGGACACGGCGGCGGTGACAGCTACGGACAAGGCGGTGGTGACAGCTACGGACATGGTGGCGGTGACAGCTACGGACATGGTGGCGGTGACTTCGGTGGCTCCAGCGGTGGCTGGAAACCCATTGCCTCTGATGAAGGCCACGGACACCATCACCACCAACACCACGAACACGTCAAGACCATCGAAGTCATCAAGAAGGTCCCTGTCCCATACACCGTTGAGAAGCACGTCCCCTACACCGTTGAGAAGAAAGTACCTTATGAAGTCAAAGTACACGTGCCCCAGCCCTACACCATCGAAAAGAAAGTCCCCGTCACCGTGAAGGAGTACGTCAAATACCCCGTGTACGTGCCTGAGCCCTACACCGTTGAGAAGAAGGTACCATATGAAGTCAAGGTCCCCGTCGACAAGCCCTACGAAGTGAAGGTTAAGGTGCCCACTCCTTACACCGTCGAAAAGAAGGTCCCCTATGAAGTCAAGGTCCCCGTACCCCAGCCCTACACTGTCGAGAAGAAGGTCCCAGTCCCCGTGAAATACGAAGTCAAAGTACCCCAACCCTACGAAGTCGTCAAGAAGGTGCCTTATGAAGTTAAAGTGCCCGTCGACAAGCCCTACAACGTGTACGTGCCCAAACCTTACCCAGTCCCTGTTGAGAAGCCCTACCCCGTCACAGTCCACAAGCCTGTGCCCTATGAAGTCAAAGTCCCCGTTGACAAGCCTTACAAGGTTGAGGTTGAGAAGCCCTACCCAGTCCACATCAAGGTCCCAGTGCCCAAACCTTACGACGTGTACAAGAAGATCCCATACACCGTTGAGAAGAAGGTGCCTTTCGAAGTGAAGGTCCCCATTGACAAGCCTTACCCAGTGTACAAGGACGTGCCATTCCCATTGGTGAAGGAGGTGCCTTACCCCGTGAAGGTGCATGTTCCCTTCTACATCAAAAAGGAGGAGGAGCACCACCACCACGAGCACCACCAGCCCCAGCAACACCACGGTTGGCACTGA
Amino acid sequence of SEQ ID No.2 tin encoded protein
mkflvvlvaslavvafaseekgapkqaaasedkkqekrgiydfghygggggsseggygghgfgggesvsfghgggesfghgggesfghgggdsygqgggdsyghgggdsyghgggdfggssggwkpiasdeghghhhhqhhehvktievikkvpvpytvekhvpytvekkvpyevkvhvpqpytiekkvpvtvkeyvkypvyvpepytvekkvpyevkvpvdkpyevkvkvptpytvekkvpyevkvpvpqpytvekkvpvpvkyevkvpqpyevvkkvpyevkvpvdkpynvyvpkpypvpvekpypvtvhkpvpyevkvpvdkpykvevekpypvhikvpvpkpydvykkipytvekkvpfevkvpidkpypvykdvpfplvkevpypvkvhvpfyikkeeehhhhehhqpqqhhgwh
SEQ ID No.11dstitin
GCAGCATCCGAAGATAAGAAGCAGGAGAAGAGAGGAATTTATGACTTTGGTCATTATGGAGGCGGTGGTGGCAGCAGCGAGGGAGGTTATGGAGGACATGGTTTCGGCGGTGGCGAAAGTGTGAGCTTCGGACACGGCGGTGGTGAAAGCTTCGGTCATGGAGGTGGCGAGAGCTTCGGACACGGCGGCGGTGACAGCTACGGACAAGGCGGTGGTGACAGCTACGGACATGGTGGCGGTGACAGCTACGGACATGGTGGCGGTGACTTCGGTGGCTCCAGCGGTGGCTGGAAACCCATTGCCTCTGATG
Extracting total RNA of prodenia litura by using a Trizol method, and synthesizing cDNA by reverse transcription. Performing PCR amplification by taking dsetin F/dsetin R as a primer and prodenia litura cDNA as a template; the PCR amplification reaction system is as follows: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 45s, total of 35 cycles, and extension at 72℃for 7min. The PCR product is dissolved in a proper amount of ddH through agarose gel electrophoresis and gel recovery 2 In O, the dsRNA was synthesized using HiScribeTMT7 In Vitro Transcription Kit using the gel-recovered PCR product as a template. Purification of dsRNA product using sodium acetate and isopropanol, solubilization in ddH 2 O, and storing at-20deg.C (see kit product instruction for specific methods). Purified dsRNA product was dissolved in RNase Free ddH 2 O, and storing at-20deg.C.
Example 2 Effect of dsetin on prodenia litura survival
Placing each 30 larvae of the dsRNA treatment group and the control group respectively in a climatic chamber with the temperature of 27-28 ℃ and the humidity of 30-40% and the photoperiod of 14 L:10D for culture, feeding fresh artificial feed, counting the death number of the larvae every day, and counting for 7 days.
Daily mortality of 2-year-old larvae after dstin injection was counted and indicated 36.67% for 7 days and 0% for control group mortality (fig. 1). The dsRNA is partially degraded in the alimentary canal, so that the prodenia litura larva does not have higher mortality after the dstin is silenced.
Example 3 RNA interference efficiency of Spodoptera litura lethal Gene tin
The template for synthesizing dsGFP was obtained by the above method using the Green Fluorescent Protein (GFP) gene as a control and the green fluorescent protein GFP plasmid purchased from the Cantonese, optimum and Biotech Co., ltd. The PCR primer dsGFP-F/dsGFP-R was designed and a T7 promoter sequence was added to its 5' -end.
dsGFP-F：TAATACGACTCACTATAGGGGACGACGGCAACTACAAGAC(SEQ ID No.5)
dsGFP-R：TAATACGACTCACTATAGGGCTTGTACAGCTCGTCCATGC(SEQ ID No.6)
After the reaction system is configured, PCR reaction is carried out, and the PCR product is dissolved in a proper amount of ddH through agarose gel electrophoresis and gel recovery 2 In O, the gel recovery PCR product is used as a template to synthesize dsRNA, and sodium acetate and isopropanol are used to purify the dsRNA product, and the dsRNA product is dissolved in ddH 2 O.
Microinjection of dsRNA: dsRNA is injected into 2-instar larvae of prodenia litura, 100nL of each larva is injected, and samples are collected after 48h to detect the effect of RNA interference on the silencing of the tin gene.
Microinjection of nanomaterial-mediated dsRNA: imidazole modified graphene nanomaterial carbon quantum dots CQDs (structure schematic diagram is shown in FIG. 3; xianfeng nanomaterial technologies Co., ltd.) are selected. The concentration of dsRNA and carbon quantum dots is 100 ng/. Mu.L. The dsRNA and the compound of different amounts of carbon quantum dots are uniformly mixed according to the volume ratio of dsRNA to CQDs of 1:0, 2:1, 3:1, 4:1, 5:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 and 20:1, the effect of combining the dsRNA with the compound of different amounts of carbon quantum dots is detected on 1% agarose gel, the migration band strength of 310bp dsRNA is gradually increased along with the increase of the volume ratio (figure 4), and when the volume ratio of dsRNA to CQDs is=11:1, the wrapping capacity of the carbon quantum dots to the dsRNA is weaker, so that the bearing capacity of the carbon quantum dots to the 310bp dsRNA is determined to exceed the limit at the ratio, and the ratio is selected for subsequent microinjection experiments. dsRNA and carbon quantum dots are mixed according to the volume ratio of 11:1 (overload), and then injected into 2-instar larvae of prodenia litura (CQDs wrap the absolute quantity of the dsRNA and only 1/10 of that of the original injected naked dsRNA), so as to ensure the consistent quality of the injected dsRNA, and the effect of RNA interference on the tin gene silencing is detected after 109nL and 48h of each insect injection.
Designing a tin gene qRT-PCR Primer by using Primer5, and carrying out qRT-PCR amplification by using prodenia litura cDNA as a template, wherein a PCR reaction system is as follows: TB Green II 5 mu L, cDNA template 2 mu L, forward and reverse primer mix 1 mu L, ddH 2 O2. Mu.L. The prodenia litura GADPH is selected as an internal reference gene.
The primer sequences were as follows:
q-titin-F：CCCTACCCAGTCCACATCA(SEQ ID No.7)
q-titin-R：AGGCACCTCCTTCACCAA(SEQ ID No.8)
GADPH-F：GGGTATTCTTGACTACAC(SEQ ID No.9)
GADPH-R：CTGGATGTACTTGATGAG(SEQ ID No.10)
analysis of variance was performed by single factor using IBM SPSS Statistics for Windows, version 19.0, multiple comparisons were performed using Tukey's method, p <0.05 was considered statistically significant.
The results showed that the relative expression level of the tigin was reduced by 2.73 times 48 hours after the dsetin injection, which was significantly different from the control group (fig. 2), and the relative expression level of the tigin gene was reduced by 27.7 times 48 hours after the dsetin injection plus carbon quantum dots, which was significantly different from the control group (fig. 5).
EXAMPLE 4 study of expression and feeding Effect of dsRNA bacterial liquids
In consideration of the fact that a large amount of dsRNA can be obtained through bacterial liquid expression, the cost is low, and the dsRNA is popularized and used in a large scale in actual life. Thus, a bacterial liquid expression vector was constructed.
The dsRNA construct was introduced into L4440 vector (Optimum Cangzhou Praeparata). The construction method of the vector comprises extracting L4440 vector plasmid from Escherichia coli, performing enzyme digestion with restriction enzymes NotI and NheI, connecting with fragment of target gene (tin), detecting, constructing the vector (constructed vector is as shown in figure 6, construction method of dsGFP is the same), taking 1mL LB liquid medium, adding 1 μL Amp (concentration 100 mg/mL) and 1 μL Tet (concentration 12.5 mg/mL), mixing uniformly, adding appropriate amount of HT115 strain bacterial liquid, sealing with sealing film, placing in shaking table, 37 ℃, culturing at 200rpm overnight. The next morning, 5mL LB liquid medium was added with 5. Mu.L Amp and 5. Mu.L Tet, and 50. Mu.L (1:100) of the overnight cultured bacterial liquid was placed on a shaking table at 37℃for 3h at 200rpm, and the bacterial liquid was cloudy. 50 μl of IPTG (concentration 100 mM) is added at a ratio of 1:100, the mixture is placed on a shaking table at 37 ℃ at 200rpm for 5 hours, a refrigerated centrifuge at 600 rpm for 4 ℃ for 5min for bacterial recovery, and bacterial liquid precipitate is frozen and stored by liquid nitrogen.
500. Mu.L of the overnight activated culture broth was transferred to 50mL of LB liquid medium containing Amp (100. Mu.g/mL) and Tet (10. Mu.g/mL), and the broth was diluted 100-fold for expansion culture. Then, 50mL of the expanded culture was transferred to 5000mL of YT liquid medium containing 5mLAmp (100. Mu.g/mL) and 5mLTet (10. Mu.g/mL), and the expanded culture was diluted 100-fold. Culturing until the OD value of the bacterial liquid is between 0.5 and 0.8. IPTG was then added to the bacterial solution to a final concentration of 1mM and cultured with shaking at 37 ℃ at 120rpm for 5h to induce dsRNA. Then, the 5000mL of the bacterial liquid was heated at 80℃for 20 minutes to kill the bacteria, and the pellet was frozen and centrifuged at 6000rpm and 4℃for 5 minutes, and the pellet was stored frozen with liquid nitrogen. Adding enzyme-free water into the sediment stored by freezing liquid nitrogen, wherein the dosage proportion of the enzyme-free water is as follows: 100. Mu.L of enzyme-free water was added to the pellet collected after centrifugation of each 15mL of the bacterial liquid. Shaking and mixing uniformly to obtain slightly thick and turbid liquid. The method is used for the prodenia litura larva feeding experiment. Starting with 1-year-old larvae, each 1-2-year-old larvae is fed with 2 mu L of bacterial liquid every day, 3-4-year is 5 mu L, 5-6-year-old larvae is 10 mu L, and a control group (dsGFP) is arranged in the same way. The number of prodenia litura deaths per dish was counted every 24h with 10 larvae 1 repeat, 5 repeats, and the mortality was calculated and the results are shown in fig. 7. The results show that the mortality rate increases significantly at day 5 and peaks at days 9-15, with a mortality rate of 58%.
Claims (10)
1. The application of the coded protein of the prodenia litura lethal gene tin in insect control is characterized in that: the amino acid sequence is shown as SEQ ID No. 2.
2. The application of the prodenia litura lethal gene tin in insect control is characterized in that: the nucleotide sequence is shown as SEQ ID No.1 or SEQ ID No. 12.
3. A method of insect control, characterized by: the main steps are to reduce the expression of gene tin in insect body.
4. A method according to claim 3, characterized in that: the RNAi technology is adopted to reduce the expression of gene tin in the insect body.
5. The method according to claim 4, wherein: in the RNAi technology, the primer sequences for amplifying dstin are shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequence of the dstin is shown as SEQ ID No. 11.
6. The method according to claim 4, wherein: injecting or/and feeding insects by using dstin; further, the insect is prodenia litura.
7. The method according to claim 6, wherein: the injection is to inject 2-year-old larvae.
8. The method according to claim 5, wherein: mixing the dstin with the carbon quantum dots and injecting; the dosage of the carbon quantum dots is overloaded.
9. A substance for reducing expression of gene tin, characterized in that: adopting RNAi technology to interfere the dstin expressed by the gene tin; the primer sequences for amplifying the dstin are shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequence of the dstin is shown as SEQ ID No. 11.
10. Use of a substance for reducing expression of the gene tin according to claim 9 for controlling insects.
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