CN116589551A - Locust immunity-related protein LmEaster gene and application thereof - Google Patents
Locust immunity-related protein LmEaster gene and application thereof Download PDFInfo
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- 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
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- 241000238631 Hexapoda Species 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
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- 238000012408 PCR amplification Methods 0.000 description 1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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.
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| CN116179608A (en) * | 2023-01-05 | 2023-05-30 | 昆明理工大学 | Application of dsRNA in construction of immunodeficiency Chinese bee model |
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|---|---|---|---|---|
| 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 |
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