CN114990228A - Green fly-killing agent - Google Patents
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- CN114990228A CN114990228A CN202210695492.5A CN202210695492A CN114990228A CN 114990228 A CN114990228 A CN 114990228A CN 202210695492 A CN202210695492 A CN 202210695492A CN 114990228 A CN114990228 A CN 114990228A
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Abstract
The invention discloses a green fly-killing agent, and belongs to the technical field of biological medicines. The green fly-killing agent has the functions of inhibiting the activation of the housefly proPO system and destroying the immunity of the housefly proPO system. The knock-out effect of musca domestica mdPAP1 and mdProPO1 genes was demonstrated by qRT-PCR technology. Enzyme activity detection proves that mdPAP1 and mdRoPO 1 gene knockout can effectively inhibit proPO system activation and reduce PO activity. Through statistics of survival rate of housefly larvae, it is proved that knocking out mdPAP1 and mdProPO1 genes promotes death of the housefly larvae. In addition, the mortality rate of the housefly larvae was found to be proportional to the mixed dsRNA dose of mdPAP1 and mdProPO1 in the housefly feed. The method can be used for obtaining related products such as housefly green insecticide and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a green fly-killing agent.
Background
The housefly is a sanitary pest in the world, has hairy body surface, can secrete mucus at the sole of a foot, is accompanied by habits of eating and vomiting, is easy to be attached with a large number of pathogens, is a carrier of various bacteria, viruses and protozoa, can transmit various diseases such as cholera, typhoid, tuberculosis, bacillary dysentery, infantile diarrhea and the like to human beings, and seriously influences the production of livestock and poultry, so that a large amount of fly-killing agents are used by countries every year to kill flies, and the consumption of the flies in the whole world reaches 7.6 billion dollars in 2021 year alone.
The fly killing agent in the current market mainly depends on chemical insecticides such as organic phosphorus, organic chlorine, pyrethroid and the like. The lack of regulatory use and even abuse of chemical pesticides has caused many problems over the years, including increased resistance to houseflies, poisoning humans with non-target organisms, environmental pollution, etc. Therefore, the research and development of the chemical insecticide substitute which has high safety to the environment and human and can effectively kill the houseflies has important practical significance and application value.
Like other insects, houseflies lack adaptive immunity and can only defend against external pathogen invasion by virtue of innate immunity. The innate immunity of insects includes both cellular and humoral immunity. The prophenoloxidase system (proPO system) is an important humoral immune component, the activation of which is a serine protease cascade: the upstream serine protease hydrolyzes zymogen of downstream enzyme, the activated enzyme inactivates zymogen of next enzyme, finally prophenoloxidase activating enzyme (PAP) directly activates prophenoloxidase (proPO) into Phenoloxidase (PO), thereby making the fastest immune response to pathogen invasion, influencing not only melanin synthesis of insects, but also development and life of the insects, being related to generation of insect antibacterial peptide, and playing an important role in pathogen recognition and immune defense reaction of the insects.
RNA interference (RNAi) technology is a new technology developed in recent years to suppress gene expression, and its principle is to suppress or even eliminate gene expression by inducing the specific degradation of homologous target gene mRNA in vivo by exogenous and endogenous double-stranded RNA (dsrna), resulting in post-transcriptional silencing of the target gene. The technology has research reports on linear animals (caenorhabditis elegans and the like), lepidoptera (tobacco hornworm and the like) and coleoptera (Holotrichia parallela and the like), and shows wide application prospects in aspects of gene functions, medicine research and development and the like.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a green fly killer. The principle of the green fly-killing agent developed by the invention is that the mdPAP1 and mdropO 1 genes of the housefly are knocked out by an RNAi technology, the activation of a housefly proPO system is inhibited, the housefly immune system is damaged, and the death of the housefly is promoted.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides application of housefly mdPAP1 and mdProPO1 genes as targets in preparing a medicament for killing houseflies.
In a second aspect of the invention, there is provided the use of an agent that inhibits the housefly mdPAP1 and mdropO 1 genes in the preparation of a medicament for killing housefly.
In the application, the reagent for inhibiting the housefly mdPAP1 and mdropo 1 genes is selected from one or more of dsRNA, small interfering RNA (siRNA) or artificial miRNA (artificial microRNA).
Preferably, the agent is dsRNA targeting the housefly mdPAP1 and mdropo 1 genes; more preferably, the sequence of dsRNA targeting the musca domestica mdPAP1 gene is shown in SEQ ID No. 7; the method comprises the following specific steps:
TGGAATGTCCTCCCTTGTTGAATCTTTTGAAAAATGTTGGTAGAACCCAAGCAGAAACCATGTTTCTGCAACACAGTCAATGCGATTATGTGGGTTCCACTGTTTATGTTTGCTGTGTTTTGCAAAGCGGTAGCCGTTTCCTAAAAGCTGAATTGCCAACAACACGCGAATGTGGCAAATCGTTCGATAATCGCATTCTTGGTGGAAATGTTACCAGAATCGATGAATATCCTTGGGTGGCATTAATCGAGTATACCAAACCCTTCAATGAAAAAGGTTTCCACTGTGGTGCAGCTCTCATCAGCAAACGTTATGTCATAACGGCGGCAC (SEQ ID NO.7) the sequence of dsRNA targeting musca domestica mdropO 1 gene is shown in SEQ ID NO. 8; the method comprises the following specific steps:
GATGGATTCACTGGTTGCCAGCCGTGCTTGGCCACCACGTTTCGATAATACTTCCATCAAAGATTTGAATCGTGAATTGGATCAAATCAATTTGGACATTTCAGACTTGGAAAGATGGCGTGATCGTATTTTCGAGGCCATCCATCAAGGATTTGTGGTCGATGCCAGCGGCAATCGTATTCCTTTGGATGAACGTCGTGGTATTGATATTCTGGGTAATATGTTGGAAGCTTCCATCATTTCACCCAATCAATCGGTGTATGGTGATTTCCATAACATGGGTCATGTCTTCATTTCCTATGCCCACGATCCTGATCATCGCCATCTGGAGTCATTCGGCGTAATGGGTGATTCAG.(SEQ ID NO.8)
in a third aspect of the invention, a green fly-killing agent is provided, wherein the fly-killing agent takes dsRNA targeting musca mdPAP1 gene and dsRNA targeting musca mdropO 1 gene as active ingredients.
Preferably, the sequence of dsRNA targeting the housefly mdPAP1 gene is shown as SEQ ID NO. 7; the sequence of dsRNA targeting the housefly mdropO 1 gene is shown in SEQ ID NO. 8.
More preferably, the weight ratio of dsRNA targeting the housefly mdPAP1 gene to dsRNA targeting the housefly mdropo 1 gene is 1: 1.
The invention has the beneficial effects that:
the green fly killer has the functions of inhibiting activation of a housefly proPO system and destroying the immunity of the housefly proPO system. Knock-out effect of musca domestica mdPAP1 and mdProPO1 genes was demonstrated by qRT-PCR technique (FIG. 1). Enzyme activity detection proves that mdPAP1 and mdropO 1 gene knockout can effectively inhibit proPO system activation and reduce PO activity (figure 2). Knock-out of mdPAP1 and mdropo 1 genes was shown to promote housefly larvae death by housefly larvae survival statistics (fig. 3). In addition, the mortality of housefly larvae was found to be proportional to the mixed dsRNA dose of mdPAP1 and mdropo 1 in the housefly feed (fig. 4). The method of the invention can be used for obtaining related products such as housefly green insecticide and the like.
Drawings
FIG. 1 shows the relative expression levels of mdPAP1 and mdProPO1 genes in knock-out houseflies; each set of data was three independent replicates, with asterisks representing significant differences from controls (. about.p <0.001,. about.p <0.01,. about.p < 0.05);
wherein, figure 1A experimental group is dsmdPAP1, representing that the feed is mdPAP1 dsRNA and normal housefly feed (bran) according to the ratio of 2:10 6 Mixing materials in proportion; FIG. 1B shows dsmdropO 1 as the experimental group, and mdropO 1 dsRNA and normal housefly feed (bran) are mixed according to the ratio of 2:10 6 Mixing materials in proportion; the control group was dsGFP, the feed was an equivalent amount of GFP dsRNA to normal housefly feed (bran) at 2:10 6 Stirring materials according to the proportion. Compared with the control group, the expression level of mdPAP1 and mdProPO1 genes in the experimental group is obviously reduced.
FIG. 2 shows the results of PO activity assays in mdPAP1 and mdropO 1 gene knock-out Musca domestica; each set of data was triplicated independently and the asterisks represent significant differences from the controls (. about.p <0.001,. about.p <0.01,. about.p < 0.05).
Wherein the control group is No. 1, the representative feed is GFP dsRNA and normal housefly feed (bran) according to the ratio of 2:10 6 And (4) stirring materials in proportion. The experimental group is No. 2, and represents the 1:1 mixed dsRNA of feed mdPAP1 and mdProPO1 and normal housefly feed (bran) at 2:10 6 Stirring materials according to the proportion. The PO activity of the housefly larvae was significantly reduced in the experimental group in which mdPAP1 and mdropo 1 genes were disrupted, compared to the control group.
FIG. 3 is the survival rate of Musca domestica larvae with mdPAP1 and mdProPO1 gene knockout; the abscissas 1-7 represent housefly larvae fed on different feeds. 1: normal feed, 2: DEPC water mix, 3: mixed bacteria mixing and stirring material of escherichia coli and staphylococcus aureus, 4: GFP dsRNA + Escherichia coli and staphylococcus aureus mixed bacteria mixing and stirring material, 5: mdPAP1 dsRNA + escherichia coli and staphylococcus aureus cocktail, 6: mdropo 1 dsRNA + escherichia coli and staphylococcus aureus mixed bacteria mixing material, 7: mixed dsRNA + escherichia coli and staphylococcus aureus dressing of mdPAP1 and mdropo 1. The ordinate is the statistical survival of housefly larvae fed on different feeds, each group of data was triplicated independently, and asterisks represent significant differences from the controls (. about.p <0.001,. about.p <0.01,. about.p < 0.05).
The newly laid eggs of normal houseflies are inoculated into the following 7 groups of feeds: normal feed, 0.8mL DEPC water mixed feed, 0.8mL Escherichia coli, and Staphylococcus aureus mixed strain (1.5X 10) 8 CFU/mL), 2. mu.g GFP dsRNA, and 0.8mL E.coli, S.aureus mixed strain (1.5X 10) 8 CFU/mL), 2. mu.g of mdPAP1 dsRNA, and 0.8mL of a mixture of E.coli and S.aureus (1.5X 10) 8 CFU/mL) blend, 6: mu.g mdropO 1 dsRNA, Escherichia coli and Staphylococcus aureus mixed strain (1.5X 10) 8 CFU/mL), 2. mu.g of 1:1 mixed dsRNA of mdPAP1 and mdropO 1 and 0.8mL of mixed Escherichia coli and Staphylococcus aureus (1.5X 10) 8 CFU/mL) was stirred. Each group of feed has 3 repetitions, each repetition has 100g of feed, and 50 housefly eggs are inoculated. After three days, the surviving housefly larvae are collected and the survival rate is counted. The results show that the survival rate of the housefly larvae of the experimental group knocked out the mdPAP1 gene and the mdropO 1 gene is obviously reduced compared with that of the other 4 control groups, and the fly killing effect of the dsRNA of the mdPAP1 gene and the dsRNA of the mdropO 1 gene mixed sample is obvious.
FIG. 4 is the mortality of Musca domestica larvae knocked out with mdPAP1 and mdropO 1 genes; dsGFP was control: feeding mixed materials of GFP dsRNA with different contents, escherichia coli and staphylococcus aureus by housefly larvae; dsmdPAP1+ dsmdProPO1 was the experimental group: the housefly larvae are fed with mixed dsRNA of different contents of mdPAP1 and mdropO 1 and mixed feed of mixed bacteria of escherichia coli and staphylococcus aureus. Each set of data was triplicated independently and the asterisks represent significant differences from the controls (. about.p <0.001,. about.p <0.01,. about.p < 0.05).
And (4) inoculating the newly-laid eggs of the normal houseflies into the feeds of the control group and the experimental group. Control group feed: GFP dsRNA containing different amounts (2 mug, 4 mug, 6 mug and 8 mug) is mixed with escherichia coli and staphylococcus aureus. Experimental group feed: mixed dsRNA containing different amounts (2. mu.g, 4. mu.g, 6. mu.g, 8. mu.g) of mdPAP1 and mdropO 1 in a ratio of 1:1, and mixed bacteria of Escherichia coli and Staphylococcus aureus. Each treatment was 3 replicates. After three days, the surviving three-instar housefly larvae from each treatment were collected and their mortality was counted. The results show that the higher the content of mdPAP1 and mdProPO1 mixed dsRNA in the feed, the higher the death rate of the houseflies.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described above, based on the current situation that the housefly green insecticide is lacking, firstly, the Unigene sequences of the key genes of the housefly proPO system mdPAP1(MF095796) and mdropo 1(AY494738) are screened through the sequencing and sequence analysis of a housefly transcriptome, and the housefly mdPAP1 and mdropo 1 genes are cloned according to the screened Unigene sequences. [ paper: dianxiang Li, Yonggli Liang, Xianwei Wang, Lei Wang, Mei Qi, Yang Yu, Yuanyu Lun. Transcriptomic analysis of Musca biomedical to removable keys genes of the prophenoxidase-activating system.G3(Bethesda). 2015; 1827-1841.Xuna Zhuang, Yuanyuan Luan, Tonghui Lv, Chengming Ren, Lei Wang, Qiang Li, Dian-Xiang Li. PAP1 activators the phenoxoxidase system against bacteria induction in Musca biomedical. Dev. Immunol.2021,124:104184 ].
The invention designs the primers for amplifying the gene interference fragments according to the cDNA sequences of Musca domestica mdPAP1 and mdropO 1 genes and a control GFP gene, and amplifies the cDNA fragments interfering with the genes by using recombinant plasmids containing the cDNA sequences of the genes as templates. dsRNA of mdPAP1, mdProPO1 and GFP gene interference fragments was synthesized using a transcription kit using cDNA fragments interfering with these genes as templates. The synthesized dsRNA of the mdPAP1 and mdropO 1 genes is mixed and stirred according to the ratio of 1:1 and fed to the first instar of the housefly as an experimental group, and the first instar of the housefly fed with the dsRNA stirred and stirred with the same amount of GFP gene is used as a control group and cultured normally for 3 days. And (3) normal culture conditions: the humidity is 60%, the temperature is 25 ℃, and the light cycle is 12L: 12D. And selecting the third instar larvae which survive three days after feeding dsRNA, extracting RNA of the third instar larvae to be converted into cDNA, and detecting the interference efficiency of the mdPAP1 and mdropO 1 genes in the experiment group by qRT-PCR. The result shows that the expression level of mdPAP1 and mdropO 1 genes in an RNAi experimental group is obviously reduced, and the mdPAP1 and mdropO 1 genes are effectively knocked out.
The invention utilizes the enzyme-labeling instrument to detect the PO activity of the hemolymph of the three-instar larvae of RNAi houseflies. Hemolymph of the surviving housefly third instar larvae after feeding mdPAP1 and mdropO 1 mixed dsRNA experimental group and GFP dsRNA control group for three days is respectively extracted, and the PO activity is calculated according to the amount of melanin formed by catalyzing L-3, 4-dihydroxyphenylalanine (L-DOPA) substrate by the housefly hemolymph. As a result, the PO activity of the housefly larvae in the RNAi experiment group was significantly reduced, indicating that the housefly in the experiment group lacks MdPAP1 and MdpropO1, the proPO system thereof cannot be effectively activated, and PO production is reduced or cannot be generated.
The invention counts the survival rate of the three-instar larvae of the RNAi housefly. The newly laid eggs of normal houseflies are inoculated into the following 7 different treated feeds: the feed comprises normal feed, DEPC water stirring material, Escherichia coli and staphylococcus aureus mixed stirring material, GFP dsRNA + Escherichia coli and staphylococcus aureus mixed stirring material, mdPAP1 dsRNA + Escherichia coli and staphylococcus aureus mixed stirring material, mdROPO 1 dsRNA + Escherichia coli and staphylococcus aureus mixed stirring material, and mixed dsRNA + Escherichia coli and staphylococcus aureus mixed stirring material of the mdPAP1 and the mdROPO 1. After conventional culture for 72h, the survival rate of the three-instar larvae of the houseflies in the 5 feeds is counted respectively. The results show that the gene mdPAP1 and mdProPO1 of the housefly larvae fed with the feed of the seventh species are knocked out, the immune system of the housefly larvae is damaged, and the survival rate of the housefly larvae infected by bacteria is obviously reduced.
The invention aims to develop a green fly killer interfering with musca domestica mdPAP1 and mdProPO1 genes. RNAi technology is utilized to interfere with housefly mdPAP1 and mdropO 1 genes, RNAi efficiency is detected through qRT-PCR, the PO activity of RNAi housefly larva hemolymph is detected through a microplate reader, and the fly killing efficiency of the fly killing agent is statistically analyzed through RNAi housefly larva survival number.
The method comprises the following steps: cloning of mdPAP1 by Musca domestica transcriptome sequencing and sequence analysismdPAP1 gene, interference primers mdPAP1-iF and mdPAP1-iR, mdPPPO 1-iF and mdPPPO 1-iR, GFP-iF and GFP-iR (the primer sequences are shown in Table 1) are designed according to the gene, a T7 promoter is connected to the 5' end of the primers, and cDNA fragments interfering the mdPAP1, mDPO 1 and GFP genes are amplified by respectively taking recombinant plasmids mdPAP1/pGEX-3H, mdproPO1/pET-30a and pIEX-GFP constructed in the laboratory as templates. Using the amplified interference cDNA fragment as a template, synthesizing dsRNA of the three genes by using a transcription kit, using DEPC water to resuspend the dsRNA, and storing at-20 ℃ for later use after the concentration is determined by a nucleic acid instrument. GFP dsRNA, dsRNA of mdPAP1 alone, dsRNA of mdropO 1 alone, dsRNA of mixed mdPAP1 and mdropO 1 were mixed with housefly feed (bran) at a ratio of 2:10 6 Mixing materials according to the mass ratio, subpackaging the mixture into control group and experimental group culture dishes, respectively 3 dishes, inoculating 50 new eggs of normal houseflies into each dish, after three days of conventional culture, selecting the survival housefly third-instar larvae of the control group and the experimental group, extracting RNA (ribonucleic acid) to be converted into cDNA (complementary deoxyribonucleic acid), detecting the expression levels of mdPAP1 and mdropO 1 genes of the control group and the experimental group housefly third-instar larvae by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and analyzing the RNA interference efficiency of the mdPAP1 and mdropO 1 target genes. Hemolymph of the experimental and control groups surviving the three-instar larvae of the houseflies was extracted and their PO activity was measured. The survival rates of the three-instar housefly larvae of the experimental group and the control group are counted, and the fly killing effect of the fly killing agent is analyzed.
TABLE 1 Gene interference primers
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions or according to the conditions recommended by the reagents company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.
Example 1: and detecting the expression level of mdPAP1 and mdProPO1 in the knock-out housefly.
The method mainly comprises the following steps:
1. synthesis of mdPAP1 Gene dsRNA
The mdPAP1 cDNA was amplified using the mdPAP1-iF and mdPAP1-iR primers. The 5' ends of these primers were ligated with a T7 promoter, and the cDNA of interfering fragments was amplified using a 1:100 recombinant expression plasmid (mdPAP1/pGEX-3H) as a template, as shown in Table 2:
table 2: PCR reaction system
Preparing the reaction system into Mix, adding the Mix into a microcentrifuge tube in equal amount respectively, and putting the microcentrifuge tube into a PCR instrument;
PCR procedure:
detecting by using 1.5% gel electrophoresis, photographing after imaging and recording the detection result.
The mdPAP1 dsRNA (the nucleotide sequence of which is shown in SEQ ID NO.7) is synthesized by adopting a TranscriptAID T7 high-yield transcription kit. The samples shown in the table below were added to a microcentrifuge tube, mixed well, and centrifuged instantaneously.
Table 3: reaction system
After 5h at 37 ℃ 8. mu.L LDNase I (1U/. mu.L) was added to the reaction tube for digestion and incubation at 37 ℃ for 30 min.
Adding 161 mu L of DEPC treated water and 21 mu L of 3M sodium acetate into the 28 mu L reaction system of the digested dsRNA, and thoroughly mixing; extracting with 1:1 water-saturated phenol chloroform, mixing, centrifuging at 12000rpm at 4 deg.C for 15 min; adding 2 times of chloroform into the supernatant, centrifuging at 4 ℃ and 12000rpm for 15min, and removing the supernatant; the resulting precipitate of mdPAP1 dsRNA was added with 2 volumes of pre-cooled absolute ethanol and then placed at-20 ℃ for 30 min. Then, the mixture is centrifuged at 12000rpm for 5min, and the supernatant is discarded; the mdPAP1 dsRNA pellet was then washed by adding 500. mu.L of pre-chilled 70% ethanol. Centrifuged at 12000rpm for 5min to remove ethanol, air-dried, and then suspended in 20. mu.L of DEPC-treated water and stored at-80 ℃. mu.L of mdPAP1 dsRNA was taken and its concentration was measured in a nucleic acid detector.
(2) Synthesis of mdpropO1 gene dsRNA
The mdropO 1 cDNA was amplified using mdropO 1-iF and mdropO 1-iR primers. The 5' ends of these primers were ligated with a T7 promoter, and cDNA of the interfering fragment was amplified using a 1:100 dilution of recombinant expression plasmid (mdropO 1/pET-30a) as a template. mDPROPO1 dsRNA synthesis procedure As described above, the nucleotide sequence of the synthesized mDPROPO1 dsRNA is shown in SEQ ID NO. 8.
(3) Synthesis of GFP Gene dsRNA
GFP cDNA was amplified using designed GFP-iF and GFP-iR primers using pIEX-GFP plasmid diluted at 1:100 as template. The GFP dsRNA synthesis steps are as described above, and the nucleotide sequence of the synthesized GFP dsRNA is shown as SEQ ID NO.9, and is as follows:
TGGTCCCAATTCTCGTGGAACTGGATGGCGATGTGAATGGGCACAAATTTTCTGTCAGCGGAGAGGGTGAAGGTGATGCCACATACGGAAAGCTCACCCTGAAATTCATCTGCACCACTGGAAAGCTCCCTGTGCCATGGCCAACACTGGTCACTACCTTCACCTATGGCGTGCAGTGCTTTTCCAGATACCCAGACCATATGAAGCAGCATGACTTTTTCAAGAGCGCCATGCCCGAGGGCTATGTGCAGGAGAGAACCATCTTTTTCAAAGATGACGGGAACTACAAGACCCGCGCTGAAGTCAAGTTCGAAGGTGACACCCTGGTGAATAGAATCGAGCTGAAGGGCATTGACTTTAAGGAGGATGGAAACATTCTCGGCCACAAGCTGGAATACAACTATAACTCCCACAATGTGTACATCATGGCCGACAAGCAAAAGAATGGCATCAAGGTCAACTTCAAGATCAGACACAACATTGAGGATGGATCCGTGCAGCTGGCCGACCATTATCAACAGAACACTCCAATCGGCGACGGCCCTGTGCTCCTCCCAGACAACCATTACCTGTCCACCCAGTCTGCCCTGTCTAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTGCTGGAGTTTGTGACCGCTGCTGGGATCACACATGGCATGGACGAGCTGTAC。(SEQ ID NO.9)
(4) randomly selecting 100 normal houseflies to newly lay eggs, and averagely dividing the normal houseflies into 2 groups: 1 experimental group, 1 control group. The experimental group feed was 1:1 (by weight) of mdPAP1 and mdropO 1Ratio) mixing dsRNA with normal housefly feed (bran) at a ratio of 2:10 6 The materials are mixed according to the weight ratio, a GFP control group is fed with GFP dsRNA and normal housefly feed (bran) according to the ratio of 2:10 6 And (4) stirring materials in proportion. Conventional culture, culture conditions: the humidity is 60%, the temperature is 25 ℃, and the photoperiod is 12L: 12D.
After 3 days, 4 surviving three-year-old housefly larvae are randomly picked from each group, RNA is extracted and inverted into cDNA, and the expression quantity of mdPAP1 and mdropO 1 in the housefly bodies is knocked out through qRT-PCR analysis.
The results are shown in FIG. 1 and show that: the expression level of mdPAP1 and mdProPO1 genes in the experimental group is obviously reduced, and the mdPAP1 and mdProPO1 genes are effectively knocked out.
Example 2: the main steps of detecting the change of PO activity in the housefly bodies of mdPAP1 and mdProPO1 gene knockout are as follows:
randomly selecting 200 normal houseflies to newly lay eggs, and dividing the houseflies into an experimental group and a control group 2, wherein each group has 100 flies. Experimental groups used 1:1 mixed dsRNA of mdPAP1 and mdropO 1 and normal housefly feed (bran) at 2:10 6 The proportion of the mixed materials is cultured, and the GFP dsRNA and normal housefly feed (bran) are used for a control group according to the proportion of 2:10 6 Mixing and culturing according to the weight ratio. And (3) conventional culture: the humidity is 60%, the temperature is 25 ℃, and the photoperiod is 12L: 12D. Three days later, 50 of the 2 groups of surviving housefly larvae were randomly picked and hemolymph was extracted for PO activity assay.
The method for preparing the hemolymph sample of the housefly larvae comprises the following steps:
the housefly larvae of the experimental group and the control group are placed in a sterilized culture dish, and the culture is washed by 0.8% of normal saline; absorbing normal saline on the surface of the insect body by using absorbent paper; clamping the larvae onto an ice-precooled sterile glass slide by using sterilized forceps; cutting off head of larva body with sterile scalpel, sucking hemolymph at incision of larva body with liquid-transferring gun, placing in precooled anticoagulant, mixing, and placing on ice; the hemolymph samples are added into a 96-well plate, and the change of housefly PO activity after the gene silencing of mdPAP1 and mdropO 1 is detected by a microplate reader.
Example 3: survival detection of Musca domestica larvae with mdPAP1 and mdProPO1 gene knocked out
The method mainly comprises the following steps:
the newly produced eggs from the housefly were collected and randomly divided into 7 groups, including 3 experimental groups and 4 control groups, each containing 50 eggs, and cultured with the following different treatments of feed.
Control group 1: normal feed (bran), 100g per dish;
control group 2: mixing 0.8mL of DEPC treated water with 100g of normal feed;
control group 3: 0.8mL of a mixture of Escherichia coli and Staphylococcus aureus (1.5X 10) 8 CFU/mL, the ratio of mixed bacteria is 1:1) and 100g of normal feed are mixed;
control group 4: mu.g GFP dsRNA and 0.8mL Escherichia coli/Staphylococcus aureus mixture (1.5X 10) 8 CFU/mL, the ratio of mixed bacteria is 1:1) and 100g of normal feed are stirred;
experimental group 1: mu.g of mdPAP1 dsRNA and 0.8mL of Escherichia coli and Staphylococcus aureus mixed strain (1.5X 10) 8 CFU/mL, the ratio of mixed bacteria is 1:1) and 100g of normal feed are mixed;
experimental group 2: mu.g mdropO 1 dsRNA and 0.8mL Escherichia coli and Staphylococcus aureus mixed strain (1.5X 10) 8 CFU/mL, the ratio of mixed bacteria is 1:1) and 100g of normal feed are mixed;
experimental group 3: 2 μ g of 1:1 Mixed dsRNA of mdPAP1 and mdropO 1 (1 μ g of mdPAP1 dsRNA and 1 μ g of mdropO 1 dsRNA in the mixed dsRNA) and 0.8mL of a mixed strain of E.coli and S.aureus (1.5X 10) 8 CFU/mL, the ratio of mixed bacteria is 1:1) and 100g of normal feed are mixed;
and (3) conventional culture: and (3) counting the survival rate of the three-instar housefly larvae after the humidity is 60%, the temperature is 25 ℃, and the light cycle is 12L: 12D.
The results are shown in FIG. 3 and show that: the survival rate of the houseflies can be obviously reduced by mixed feeding of the mdPAP1 dsRNA and the mdropO 1 dsRNA (the average survival rate of the mixed feeding is only 12.3 percent), and the feed has obvious synergistic effect compared with the single feeding of the mdPAP1 dsRNA and the single feeding of the mdropO 1 dsRNA.
Example 4: musca domestica larva mortality detection method by knocking out mdPAP1 and mdropO 1 genes
The method mainly comprises the following steps:
newly-produced eggs from normal houseflies were collected and inoculated into the following feeds for the control and experimental groups (Table 4). Control group: dressings containing different amounts (2. mu.g, 4. mu.g, 6. mu.g, 8. mu.g) of GFP dsRNA; experimental groups: mixes of 1:1 mixed dsRNA containing different amounts (2. mu.g, 4. mu.g, 6. mu.g, 8. mu.g) of mdPAP1, mdProPO 1. Each group had 3 replicates, each replicate having 50 eggs. After three days, the surviving three-instar housefly larvae from each treatment were collected and their mortality was counted.
Control group: different amounts of GFP dsRNA and 0.8ml of mixed Escherichia coli and Staphylococcus aureus (1.5X 10) were fed according to Table 4 8 CFU/mL) and 100g of bran;
experimental groups: different amounts of mdPAP1 and mdropO 1 were fed as per Table 4 with mixed dsRNA and E.coli at 1:1, mixed Staphylococcus aureus in 0.8ml (1.5X 10) 8 CFU/mL) and 100g bran.
Table 4: housefly larva mortality detection
The results are shown in FIG. 4 and show that: the higher the content of mdPAP1 and mdProPO1 mixed dsRNA in the feed, the higher the death rate of the houseflies.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Jinan
<120> a green fly-killing agent
<130> 2022
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 40
<212> DNA
<213> mdPAP1-iF
<400> 1
gcgtaatacg actcactata ggttggaatg tcctcccttg 40
<210> 2
<211> 38
<212> DNA
<213> mdPAP1-iR
<400> 2
gcgtaatacg actcactata ggtgtgccgc cgttatga 38
<210> 3
<211> 40
<212> DNA
<213> mdproPO1-iF
<400> 3
gcgtaatacg actcactata gggatggatt cactggttgc 40
<210> 4
<211> 40
<212> DNA
<213> mdproPO1-iR
<400> 4
gcgtaatacg actcactata ggctgaatca cccattacgc 40
<210> 5
<211> 43
<212> DNA
<213> GFP-iF
<400> 5
gcgtaatacg actcactata ggtggtccca attctcgtgg aac 43
<210> 6
<211> 43
<212> DNA
<213> GFP-iR
<400> 6
gcgtaatacg actcactata ggcttgaagt tgaccttgat gcc 43
<210> 7
<211> 330
<212> DNA
<213> dsRNA targeting the Musca domestica mdPAP1 gene
<400> 7
tggaatgtcc tcccttgttg aatcttttga aaaatgttgg tagaacccaa gcagaaacca 60
tgtttctgca acacagtcaa tgcgattatg tgggttccac tgtttatgtt tgctgtgttt 120
tgcaaagcgg tagccgtttc ctaaaagctg aattgccaac aacacgcgaa tgtggcaaat 180
cgttcgataa tcgcattctt ggtggaaatg ttaccagaat cgatgaatat ccttgggtgg 240
cattaatcga gtataccaaa cccttcaatg aaaaaggttt ccactgtggt gcagctctca 300
tcagcaaacg ttatgtcata acggcggcac 330
<210> 8
<211> 356
<212> DNA
<213> dsRNA targeting musca domestica mdropO 1 gene
<400> 8
gatggattca ctggttgcca gccgtgcttg gccaccacgt ttcgataata cttccatcaa 60
agatttgaat cgtgaattgg atcaaatcaa tttggacatt tcagacttgg aaagatggcg 120
tgatcgtatt ttcgaggcca tccatcaagg atttgtggtc gatgccagcg gcaatcgtat 180
tcctttggat gaacgtcgtg gtattgatat tctgggtaat atgttggaag cttccatcat 240
ttcacccaat caatcggtgt atggtgattt ccataacatg ggtcatgtct tcatttccta 300
tgcccacgat cctgatcatc gccatctgga gtcattcggc gtaatgggtg attcag 356
<210> 9
<211> 680
<212> DNA
<213> GFP dsRNA
<400> 9
tggtcccaat tctcgtggaa ctggatggcg atgtgaatgg gcacaaattt tctgtcagcg 60
gagagggtga aggtgatgcc acatacggaa agctcaccct gaaattcatc tgcaccactg 120
gaaagctccc tgtgccatgg ccaacactgg tcactacctt cacctatggc gtgcagtgct 180
tttccagata cccagaccat atgaagcagc atgacttttt caagagcgcc atgcccgagg 240
gctatgtgca ggagagaacc atctttttca aagatgacgg gaactacaag acccgcgctg 300
aagtcaagtt cgaaggtgac accctggtga atagaatcga gctgaagggc attgacttta 360
aggaggatgg aaacattctc ggccacaagc tggaatacaa ctataactcc cacaatgtgt 420
acatcatggc cgacaagcaa aagaatggca tcaaggtcaa cttcaagatc agacacaaca 480
ttgaggatgg atccgtgcag ctggccgacc attatcaaca gaacactcca atcggcgacg 540
gccctgtgct cctcccagac aaccattacc tgtccaccca gtctgccctg tctaaagatc 600
ccaacgaaaa gagagaccac atggtcctgc tggagtttgt gaccgctgct gggatcacac 660
atggcatgga cgagctgtac 680
Claims (6)
1. The application of housefly mdPAP1 and mdropO 1 genes as targets in preparing medicaments for killing houseflies.
2. The application of the reagent for inhibiting the musca domestica mdPAP1 and mdropO 1 genes in preparing the medicament for killing the musca domestica.
3. The use according to claim 2, wherein the agent inhibiting the housefly mdPAP1 and mdProPO1 genes is selected from one or more of dsRNA, siRNA or artificial miRNA.
4. The use of claim 3, wherein the agent is dsRNA targeting the housefly mdPAP1 and mdProP O1 genes.
5. The use of claim 4, wherein the dsRNA targeting the Musca domestica mdPAP1 gene has the sequence shown in SEQ ID No. 7; the sequence of dsRNA targeting the housefly mdProPO1 gene is shown in SEQ ID NO. 8.
6. A green fly-killing agent is characterized in that the fly-killing agent takes dsRNA targeting musca domestica mdPAP1 gene and dsRNA targeting musca domestica mdropO 1 gene as active ingredients;
the sequence of dsRNA targeting the housefly mdPAP1 gene is shown as SEQ ID NO. 7; the sequence of dsRNA targeting the housefly mdProPO1 gene is shown in SEQ ID NO. 8.
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Non-Patent Citations (2)
| Title |
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| XU-NA ZHUANG等: "PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica", 《DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY》, no. 124, 23 June 2021 (2021-06-23), pages 7 * |
| 于洋: "家蝇酚氧化酶原免疫制剂的制备", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 3, 15 March 2017 (2017-03-15), pages 12 * |
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