CN116589551A - Locust immunity-related protein LmEaster gene and application thereof - Google Patents
Locust immunity-related protein LmEaster gene and application thereof Download PDFInfo
- Publication number
- CN116589551A CN116589551A CN202310033208.2A CN202310033208A CN116589551A CN 116589551 A CN116589551 A CN 116589551A CN 202310033208 A CN202310033208 A CN 202310033208A CN 116589551 A CN116589551 A CN 116589551A
- Authority
- CN
- China
- Prior art keywords
- locust
- lmeaster
- gene
- application
- related protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 56
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 14
- 230000036039 immunity Effects 0.000 title claims abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000002452 interceptive effect Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 150000007523 nucleic acids Chemical group 0.000 claims 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 14
- 102000040650 (ribonucleotides)n+m Human genes 0.000 abstract description 11
- 239000003910 polypeptide antibiotic agent Substances 0.000 abstract description 7
- 230000008859 change Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 4
- 238000002347 injection Methods 0.000 abstract description 2
- 239000007924 injection Substances 0.000 abstract description 2
- 241000238814 Orthoptera Species 0.000 description 8
- 230000007124 immune defense Effects 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 241000238631 Hexapoda Species 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 4
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 108091030071 RNAI Proteins 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004727 humoral immunity Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 241000223250 Metarhizium anisopliae Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000382353 Pupa Species 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002468 fat body Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
- A01K2217/058—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Insects & Arthropods (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technical field of agricultural biology, in particular to a locust immunity related protein LmEaster gene and application thereof. The application clones LmEaster gene and synthesizes double-stranded RNA, processes locust by injection method, detects the death rate of each processed locust, and the expression change of related antibacterial peptide, and proves that after the LmEaster gene is interfered, the expression of the antibacterial peptide of the locust is increased, the immune defensive ability of the locust is increased, and the death rate is further reduced.
Description
Technical Field
The application relates to the technical field of agricultural biology, in particular to a locust immunity related protein LmEaster gene and application thereof.
Background
Insects are the most widely distributed organisms on the earth and play an important role in the ecosystem. Insects are so flourishing that they are associated with their unique natural immune defenses. The influence of external factors on insects to promote the generation of antimicrobial peptides in fat bodies in bodies is a main characteristic of humoral immunity, and external induction factors comprise pathogenic microorganisms, bacteria, viruses, rays, ultrasonic waves and the like. Insect humoral immunity is mainly composed of three cascade reactions of IMD, toll and JAK/STAT. Wherein Toll pathways play an important role in insect immune regulation. Easter is located upstream of Toll pathway and is activated after the insect recognizes the microorganism, thereby activating downstreamBinding to Toll receptors, through a series of immune defensive signaling, ultimately producing antimicrobial peptides to cope with microbial infections.
Based on natural biological pesticides, the method is increasingly a reliable, green and durable control method, in particular to metarhizium anisopliae, which has proved to be very effective in controlling locusts, but cannot overcome the defects of low death efficiency, long death time and the like, mainly because of strong immune defense response of the locusts.
Disclosure of Invention
The application aims to provide a coding sequence of locust immune related protein LmEaster gene.
It is still another object of the present application to provide the use of the above-mentioned potent interfering sequences of the locust LmEaster gene.
It is still another object of the present application to provide a method for effectively controlling locust.
The locust immune related protein LmEaster gene provided by the application has the coding amino acid sequence shown in SEQ ID NO:1, and a protein shown in the formula 1.
SEQ ID NO:1
MQRVSVLSLA FLGALLAVGC RAKESDFKVE KLFVPDECTE KSKTGDMLTM HYRGTLDDGK 60
QFDSSHDRDQ PFSFQLGVGQ VIKGWDQGLV DMCVGERRRL VIPPELAYGD RGAGAVIPGG 120ATLTFEVELL SIGDSPPPVN VFKEIDADKD QNLSRDEVSE YLKKQMVAAE EGGDSEEVKR 180MLEDHDKLVD EIFQHEDKDK NGLISHDEFS GPKHDEL 217。
The locust immune related protein LmEaster gene has a nucleotide sequence shown in SEQ ID NO: 2.
SEQ ID NO:2:
atggtgaaag aaaatttaat taaatggatt gcatacatag ctacagttct gcatgtttgt 60
tgtgcacaag tatatcgctg tcacgctcca tacacatgca aaaatctagc agaatgtgtg 120
gatttattga agacaccaga gcagcatctt ttcctaccat gttcaactgg tgataacatc 180
aaagtgtgct gcccacgtcc agacaatctt ttgaggcata ataattttca tttattgaat 240
ggcacatgtg gagagttatc agatataaac agaataatat ctggtcagaa tgctacttta 300
ggacaattcc catggatggc acttctggga tatgaaggta gaaccccagg ttcaactgtc 360
ttctcatgtg gaggaactat tattaacaag agatacatct taacagctgc tcactgtgtt 420
gctactgaaa accagaaatt gaaaactgtt cgtttaggtg agctaaattt gtctacaaat 480
cctgactgtg aggattattg tgctgatcct gtggttgaaa gagatgttga aatgattgtg 540
atccatccaa agtacaataa tccatttcgc aaaaatgata tagccttggt cagggtcagt 600
tcggatatcc catatacaga ttttataagg ccaatctgcc ttccatttga taattcaaat 660
gacaccaaac gccaaatcaa atatgagatt gcagggtggg gcaaacagga ttcattggac 720
acaaatggaa gcactatact ccagtttgca actgtaactg taacaccaat tgaggaatgc 780
agtcctttgc agtcaaggca agttcagcca ttatcaaagg ttacacaagt gtgtgcaggg 840
ggacaaggac ctgatgcatg caatggtgat agtggtggtc cacttatgaa atactcgaat 900
gactcaagac cagtacaaca agttggcatt gtttcatttc gaacaggaac tgagtgtgga 960
ccaacagcaa gtgtttatac aagagttgaa ggttttcttg aatggatact tgataatatt 1020
agaccataa 1029
The effective interference sequence of the locust LmEaster gene is shown in SEQ ID NO: 3.
SEQ ID NO:3:
ttcccatgga tggcacttct gggatatgaa ggtagaaccc caggttcaac tgtcttctca 60
tgtggaggaa ctattattaa caagagatac atcttaacag ctgctcactg tgttgctact 120
gaaaaccaga aattgaaaac tgttcgttta ggtgagctaa atttgtctac aaatcctgac 180
tgtgaggatt attgtgctga tcctgtggtt gaaagagatg ttgaaatgat tgtgatccat 240
ccaaagtaca ataatccatt tcgcaaaaat gata 274
The application provides application of the effective interference sequence of the locust LmEaster gene in locust control. Through the interference sequence of the LmEaster gene ingested by the locust, the expression of the LmEaster gene is inhibited, so that the immune defense of the locust is improved, the effect of the LmEaster in the immune defense process of the locust is studied, and the purpose of controlling the locust is achieved according to the effect. The application provides a method for preventing and controlling locust, which comprises the following steps of using a nucleotide sequence shown as SEQ ID NO:3 interfering the locust LmEaster gene by the dsRNA sequence.
The application clones LmEaster gene and synthesizes double-stranded RNA, processes locust by injection method, detects the death rate of each processed locust, and the expression change of related antibacterial peptide, and proves that after the LmEaster gene is interfered, the expression of the antibacterial peptide of the locust is increased, the immune defensive ability of the locust is increased, and the death rate is further reduced.
Drawings
FIG. 1 shows the interference efficiency of the interference sequence dsLmEaster-1;
FIG. 2 shows the interference efficiency of the interference sequence dsLmEaster-2;
FIG. 3 shows the interference efficiency of the interference sequence dsLmEaster-3;
FIG. 4 shows the change in the expression level of the antibacterial peptide after the interference of locust LmEaster;
FIG. 5 shows the mortality change of the locust after interference with the locust LmEaster.
Detailed Description
The locust purified population is hatched at the temperature of 30+/-2 ℃ and the relative humidity of 60+/-5% in an artificial climate incubator, and after the locust purified population is hatched at the photoperiod of 14L:10D, the locust pupa hatched at the same time is transferred into a 60cm multiplied by 50cm multiplied by 70cm standard worm raising cage for raising, and the photoperiod of 14L:10D is hatched at the temperature of 30+/-2 ℃.
The basket (length×width×height=30 cm×12cm×9 cm), glass cover (length×width=36 cm×16 cm), and tweezers were used.
EXAMPLE 1 cloning of the LmEaster Gene
The LmEaster gene is cloned in adult locust, total RNA of locust is extracted, and cDNA is synthesized through reverse transcription. PCR was performed using cDNA as a template and LmEaster-F (5'-TGGCAACACATTACGCAG-3') and LmEaster-R (5'-CTCCATCATACTTGGGGTG-3') as primers, to amplify the LmEaster gene. The sequence of the LmEaster gene is shown as SEQ ID NO. 2.
2. Synthesis of dsRNA
dsRNA was further synthesized based on LmEaster gene. The RNAi primers of the LmEaster gene and RNAi primers of the control GFP gene were designed and the primer sequences are shown in Table 1. The cDNA was used as a template for PCR amplification to obtain a target fragment, which was stored at 4 ℃. The resulting purified product was synthesized as double stranded RNA of LmEaster and GFP according to the kit instructions of T7 riboMAX system (Promega, madison, wis., USA). And the concentration of dsLmEaster was measured with a NanoPhotometer micro-spectrophotometer, and the concentration of the solution of dsLmEaster and dsGFP was adjusted to 1000 ng/. Mu.L and kept at-20℃until use.
TABLE 1dsRNA primer sequences
The dsLmEaster-1 gene sequence is shown in SEQ ID NO:3 is shown in the figure
1TTCCCATGGATGGCACTTCT GGGATATGAAGGTAGAACCC CAGGTTCAAC TGTCTTCTCA
61TGTGGAGGAACTATTATTAACAAGAGATACATCTTAACAG CTGCTCACTG TGTTGCTACT
121GAAAACCAGAAATTGAAAAC TGTTCGTTTAGGTGAGCTAAATTTGTCTAC AAATCCTGAC
181TGTGAGGATTATTGTGCTGATCCTGTGGTT GAAAGAGATGTTGAAATGAT TGTGATCCAT
241CCAAAGTACAATAATCCATT TCGCAAAAAT GATA。
The dsLmEaster gene sequence is shown as SEQ ID NO:4 is shown in the figure
1CCCACGTCCAGACAATCTTT TGAGGCATAATAATTTTCATTTATTGAATG GCACATGTGG
61AGAGTTATCAGATATAAACAGAATAATATC TGGTCAGAAT GCTACTTTAG GACAATTCCC
121ATGGATGGCACTTCTGGGATATGAAGGTAGAACCCCAGGTTCAACTGTCT TCTCATGTGG
181AGGAACTATTATTAACAAGAGATACATCTTAACAGCTGCT CACTGTGTTG CTACTGAAAA
241CCAGAAATTGAAAACTGTTC GTTTAGGTGAGCTAAATTTGTCTACAAATC CTGACTGTGA
301GGATTATTGT GCTGATCCTGTGGTTGAAAGAGATGTTGAAATGATTGTGA TCCATCCAAA
361GTACAATAAT CCATTTCGCAAAAATGATATAGCCTTGGTCAGGGTCAGTT CGGATA。
The dsLmEaster gene sequence is shown as SEQ ID NO:5 is shown in the figure
1GGTCAGGGTCAGTTCGGATATCCCATATACAGATTTTATAAGGCCAATCT GCCTTCCATT
61TGATAATTCAAATGACACCAAACGCCAAATCAAATATGAGATTGCAGGGT GGGGCAAACA
121GGATTCATTG GACACAAATG GAAGCACTATACTCCAGTTT GCAACTGTAA CTGTAACACC
181AATTGAGGAATGCAGTCCTT TGCAGTCAAG GCAAGTTCAGCCATTATCAA AGGTTACACA
241AGTGTGTGCAGGGGGACAAG GACCTGATGCATGCAATGGT GATAGTGGTG GTCCACTTAT
301GAAATACTCGAATGACTCAAGACCAGTACAACAAGTTGGCATTGTTTCAT TTCGA。
Determination of expression level of LmEaster Gene after RNA interference
The expression level of the LmEaster gene of the locust after the interference treatment was detected. The experiment adopts double-stranded RNA respectively injected with LmEaster in groups, three-age locusts with consistent development are taken and divided into two groups for treatment, one group is an interference group, the other group is a control group, 5 mu L of dsRNA solution is injected into each locusts, after 24 hours after the locusts are treated, the whole locusts are placed in a homogenizer, 500 mu L of trizol is added, after homogenization is carried out for 2 minutes, RNA is extracted, and the cDNA is reversed according to a reverse kit. The designed fluorescent quantitative primer of LmEaster and the action gene are used as a contrast, and the real-time fluorescent quantitative PCR method is adopted to detect the expression quantity of the LmEaster genes of each treatment group. The total system of the fluorescent quantitative reaction was 20. Mu.L, cDNA template 2. Mu.L and ddH 2 O6. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.mlo/L), 2X TB Green Premix Ex Taq (Tli RNaseH Plus) 10. Mu.L each; each sample was subjected to 3 biological replicates using a 2-fold protocol ΔΔCt The relative expression amount was analyzed by the method.
The dsLmEaster RNA was injected into the locusts, and the expression level of the LmEaster gene was changed after 24 hours of each treatment, and as shown in FIGS. 1 to 3, the expression level of the locusts was significantly decreased after the interference of the LmEaster gene. Where dsLmEaster-1 has an interference efficiency of 91.15% (FIG. 1), dsLmEaster-2 has an interference efficiency of 70.13% (FIG. 2), and dsLmEaster has an interference efficiency of 83.13% (FIG. 3), so dsLmEaster-1 has the highest interference efficiency, which is selected for subsequent experimental arrangement.
Effect of dsLmEaster on locust immune defenses
The 3-year old locust, which had been starved for 12 hours in advance, was placed in sterile biological test baskets with 30 heads each in 1 repetition, and 3 treatment baskets (3 repetitions) each were fed with fresh wheat seedlings to the end of the experiment. The test was carried out indoors at 30℃and 16 L:8D.
After the double-stranded RNA injected with LmEaster was assayed for 24 hours, the amount of change in the antimicrobial peptide gene, the fluorescent quantitative primers (qPCR-safensin-F: 5'-CAGCACTTCTCCTAGCCCTT-3'; qPCR-safensin-R: 5'-TTGTAGCCCTTGTTCATGGC-3') were designed, and the expression levels of the LmEaster genes in each treatment group were detected by a real-time fluorescent quantitative PCR method. The total system of the fluorescent quantitative reaction was 20. Mu.L, cDNA template 2. Mu.L and ddH 2 O6. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.mlo/L), 2X TB Green Premix Ex Taq (Tli RNaseH Plus) 10. Mu.L each; each sample was subjected to 3 biological replicates using a 2-fold protocol ΔΔCt The relative expression amount was analyzed by the method. Death and survival were recorded daily at the same time, and 15-day locust survival rate was calculated.
The experimental results show that after dsLmEaster only, the expression level of LmEaster is reduced, and the expression level of locust antibacterial peptide safensin is increased (figure 4), which shows that the immune defensive ability of locust is enhanced; and the survival rate of the locust also increased significantly (fig. 5).
The application clones LmEaster gene and synthesizes double-stranded RNA, treats locust by injecting dsRNA, and detects the survival rate of locust between treatments, which shows that LmEaster participates in the inhibition of locust immune defense and plays an important role in the locust immune defense process.
The above embodiments are only for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (4)
1. The locust immune related protein LmEaster gene is characterized in that the gene codes for an amino acid sequence shown in SEQ ID NO:1, and a protein shown in the formula 1.
2. The locust immune related protein LmEaster gene according to claim 1, characterized in that the nucleotide sequence of the gene is as set forth in SEQ ID NO: 2.
3. A method for improving the immunity of a locust, comprising the step of feeding the locust with the interfering sequence of the locust immune related protein LmEaster gene according to claim 1, wherein the nucleic acid sequence of the interfering sequence of the locust immune related protein LmEaster gene is as shown in SEQ ID NO: 3.
4. Use of the locust immune related protein LmEaster gene of claim 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310033208.2A CN116589551A (en) | 2023-01-10 | 2023-01-10 | Locust immunity-related protein LmEaster gene and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310033208.2A CN116589551A (en) | 2023-01-10 | 2023-01-10 | Locust immunity-related protein LmEaster gene and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116589551A true CN116589551A (en) | 2023-08-15 |
Family
ID=87597831
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310033208.2A Pending CN116589551A (en) | 2023-01-10 | 2023-01-10 | Locust immunity-related protein LmEaster gene and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116589551A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116179608A (en) * | 2023-01-05 | 2023-05-30 | 昆明理工大学 | Application of dsRNA in construction of immunodeficiency Chinese bee model |
-
2023
- 2023-01-10 CN CN202310033208.2A patent/CN116589551A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116179608A (en) * | 2023-01-05 | 2023-05-30 | 昆明理工大学 | Application of dsRNA in construction of immunodeficiency Chinese bee model |
CN116179608B (en) * | 2023-01-05 | 2024-03-01 | 昆明理工大学 | Application of dsRNA in construction of immunodeficiency Chinese bee model |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108893472B (en) | A kind of dsRNA and its application on prevention and treatment Groton bug | |
CN103201385A (en) | Down-regulating gene expression in insect pests | |
US20230210106A1 (en) | RNAi FOR CONTROL OF PHLOEM SAP-FEEDING INSECTS IN CROP PLANTS | |
CN116589551A (en) | Locust immunity-related protein LmEaster gene and application thereof | |
CN114921467A (en) | dsRNA (double-stranded ribonucleic acid) based on myzus persicae effect factor MpC002 gene and application thereof in prevention and treatment of myzus persicae | |
CN114957426A (en) | SP6RARS and application thereof in preventing and controlling blattaria insects | |
CN110551730B (en) | Ladybug RPS18 gene and application thereof in pest control | |
CN116970623A (en) | Gene segment for preventing and controlling wheat stem-based rot and application thereof | |
CN109943570B (en) | Bemisia tabaci salivary ferritin gene BtFer1 and application thereof | |
CN114478735A (en) | Aphid high-lethal gene and application thereof in aphid prevention and treatment | |
CN117230065A (en) | dsRNA of aphid FKBP12 gene, and preparation method and application thereof | |
CN113621620B (en) | Gene with function of regulating and controlling immunity of plutella xylostella and preparation method and application thereof | |
CN110592044B (en) | Protein kinase Fused coding gene and application thereof in preventing and treating diamond back moth | |
CN116143906A (en) | LMSerpinb1 gene and application thereof | |
CN105112423B (en) | It is a kind of enhancing mulberry tree disease resistance miRNA clone and its application | |
CN112195178B (en) | Tomato late blight-resistant long-chain non-coding RNA-lncRNA40787, cloning method and application method thereof | |
CN113789329A (en) | RNA preparation for inhibiting boring behavior of chilo suppressalis | |
CN115011577B (en) | Insect m 6 A-methylation transferase METTL3 gene fragment, dsRNA thereof and application thereof | |
CN115029357B (en) | Insect m 6 dsRNA of A methylation reading protein elF3-S6 gene and application thereof | |
CN117210462A (en) | dsRNA of insect cyclophilin gene, and preparation method and application thereof | |
CN103710352A (en) | Asiatic corn borer OfAK (focal Adhesion Kinase) gene and coding protein thereof as well as method for constructing OfAK gene RNAi (Ribose Nucleic Acid interference) engineering strain | |
CN111378665B (en) | Interference sequence of serpin5 gene of locusta migratoria manilensis and application thereof | |
CN113881679B (en) | miR-71-5 mimic for enhancing termite killing effect of metarhizium anisopliae | |
CN109161602B (en) | Primer and method for detecting transcription level of JHE gene of agasicles hygrophila | |
CN111647599B (en) | Interference sequence of locusta migratoria serpin1 gene and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |