Locusta migratoria immune-related protein LmEaster gene and application thereof

Abstract

The present application relates to the field of agricultural biotechnology, in particular to locust immune related protein LmEaster gene and application thereof. The present application clones LmEaster gene and synthesizes double-stranded RNA, adopts injection method to treat locusts, and detects the mortality of locusts in each treatment and the expression amount change of related antibacterial peptide, and confirms that after interfering with LmEaster gene of locusts, the expression amount of antibacterial peptide of locusts is increased, the immune defense ability of locusts is increased, and then the mortality is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a locust immune-related protein LmEaster gene and application thereof. BACKGROUND

[0002] Insects are the most widely distributed organisms on earth, playing an important role in the ecosystem. The reason why insects are so prosperous is related to their unique natural immune defense response. The main feature of humoral immunity is that the fat body in the body produces antimicrobial peptides under the influence of external factors such as pathogenic microorganisms, bacteria, viruses, radiation, and ultrasonic waves. Humoral immunity of insects is mainly composed of IMD, Toll and JAK / STAT three cascades. Among them, the Toll pathway plays an important role in the regulation of insect immunity. Easter is located in the upstream of the Toll pathway, which is activated after the insect recognizes the microorganism, and then activates the downstream Spätzle and Toll receptor binding, and a series of immune defense signal transduction, and finally produces antibacterial peptides to cope with microbial infection.

[0003] Natural-based biopesticides are increasingly becoming a reliable, green and sustainable control method, especially Metarhizium anisopliae, which has been proven to be very effective in controlling locusts, but cannot overcome the drawbacks of low mortality efficiency and long mortality time, mainly due to the strong immune defense response of locusts. SUMMARY

[0004] The purpose of the present application is to provide the coding sequence of the locust immune-related protein LmEaster gene.

[0005] Another purpose of the present application is to provide the application of the effective interference sequence of the above-mentioned locust LmEaster gene.

[0006] Another purpose of the present application is to provide a method for effectively preventing and treating locusts.

[0007] The locust immune-related protein LmEaster gene according to the present application codes a protein with an amino acid sequence as shown in SEQ ID NO: 1.

[0008] SEQ ID NO: 1

[0009] 1 MVKENLIKWI AYIATVLHVC CAQVYRCHAP YTCKNLAECV DLLKTPEQHL FLPCSTGDNI

[0010] 61 KVCCPRPDNL LRHNNFHLLN GTCGELSDIN RIISGQNATL GQFPWMALLG YEGRTPGSTV

[0011] 121 FSCGGTIINK RYILTAAHCV ATENQKLKTV RLGELNLSTN PDCEDYCADP VVERDVEMIV

[0012] 181 IHPKYNNPFR KNDIALVRVS SDIPYTDFIR PICLPFDNSN DTKRQIKYEI AGWGKQDSLD

[0013] 241 TNGSTILQFA TVTVTPIEEC SPLQSRQVQP LSKVTQVCAG GQGPDACNGD SGGPLMKYSN

[0014] 301 DSRPVQQVGI VSFRTGTECG PTASVYTRVE GFLEWILDNI RP.

[0015] The locust immune-related protein LmEaster gene according to the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0016] SEQ ID NO: 2:

[0017] atggtgaaag aaaatttaat taaatggatt gcatacatag ctacagttct gcatgtttgt 60

[0018] tgtgcacaag tatatcgctg tcacgctcca tacacatgca aaaatctagc agaatgtgtg 120

[0019] gatttattga agacaccaga gcagcatctt ttcctaccat gttcaactgg tgataacatc 180

[0020] aaagtgtgct gcccacgtcc agacaatctt ttgaggcata ataattttca tttattgaat 240

[0021] ggcacatgtg gagagttatc agatataaac agaataatat ctggtcagaa tgctacttta 300

[0022] ggacaattcc catggatggc acttctggga tatgaaggta gaaccccagg ttcaactgtc

[0023] ttctcatgtg gaggaactat tattaacaag agatacatct taacagctgc tcactgtgtt

[0024] gctactgaaa accagaaatt gaaaactgtt cgtttaggtg agctaaattt gtctacaaat

[0025] cctgactgtg aggattattg tgctgatcct gtggttgaaa gagatgttga aatgattgtg 540

[0026] atccatccaa agtacaata tccatttcgc aaaaatgata tagccttggt cagggtcagt

[0027] tcggatatcc catatacaga ttttataagg ccaatctgcc ttccatttga taattcaaat

[0028] 720. gcaacaac gccaaatcaa atatgagatt gcagggtggg gcaaacagga ttcattggac

[0029] acaaatgga gcactatact ccagtttgca actgtaactg taacaccaat tgaggaatgc

[0030] agtcctttgc agtcaaggca agttcagcca ttatcaaagg ttacacaagt gtgtgcaggg 840

[0031] ggacaaggac ctgatgcatg caatggtgat agtggtggtc cacttatgaa atactcgaat

[0032] gactcaagac cagtacaaca agttggcatt gtttcatttc gaacaggaac tgagtgtgga 960

[0033] ccaacagcaa gtgtttatac aagagttgaa ggttttcttg aatggatact tgataatatt1020

[0034] agaccataa 1029

[0035] The effective interference sequence of the locust LmEaster gene according to the present invention has the nucleic acid sequence shown in SEQ ID NO: 3.

[0036] SEQ ID NO: 3:

[0037] ttcccatgga tggcacttct gggatatgaa ggtagaaccc caggttcaac tgtcttctca 60

[0038] tgtggaggaa ctattattaa caagagatac atcttaacag ctgctcactg tgttgctact 120

[0039] gaaaaccaga aattgaaaac tgttcgttta ggtgagctaa atttgtctac aaatcctgac 180

[0040] tgtgaggatt attgtgctga tcctgtggtt gaaagagatg ttgaaatgat tgtgatccat 240

[0041] ccaaagtaca ataatccatt tcgcaaaaat gata 274

[0042] This invention provides an effective interference sequence for the LmEaster gene of locusts for locust control. By ingesting the interference sequence of the LmEaster gene into locusts, the expression of the LmEaster gene is inhibited, thereby enhancing the locusts' immune defense. The role of LmEaster in the locusts' immune defense process was studied, and the purpose of locust control was achieved accordingly. This invention also provides a method for locust control, which includes the step of interfering with the LmEaster gene of locusts using a dsRNA sequence with a nucleotide sequence as shown in SEQ ID NO: 3.

[0043] This invention clones the LmEaster gene and synthesizes double-stranded RNA, then treats locusts using an injection method. The mortality rate of locusts in each treatment and the changes in the expression levels of related antimicrobial peptides are detected. This confirms that interfering with the LmEaster gene in locusts increases the expression levels of antimicrobial peptides, enhances the locusts' immune defense capabilities, and consequently reduces the mortality rate. Attached Figure Description

[0044] Figure 1 Show the interference efficiency of the interference sequence dsLmEaster-1;

[0045] Figure 2 Showing the interference efficiency of the interference sequence dsLmEaster-2;

[0046] Figure 3 The interference efficiency of the interference sequence dsLmEaster-3 is shown;

[0047] Figure 4 The changes in the expression levels of antimicrobial peptides after interfering with the LmEaster locust were shown.

[0048] Figure 5 This shows the change in locust mortality rate after LmEaster was disturbed. Detailed Implementation

[0049] The purified locust population was incubated in an artificial climate incubator at a temperature of (30±2)℃, a relative humidity of (60±5)%, and a photoperiod of 14L:10D. After incubation, the locust pupae that hatched at the same time were transferred to 60cm×50cm×70cm insect rearing cages for rearing at a photoperiod of 14L:10D and a temperature of (30±2)℃.

[0050] Test basket (length × width × height = 30cm × 12cm × 9cm), glass cover plate (length × width = 36cm × 16cm), tweezers.

[0051] Example 1: Cloning the LmEaster gene

[0052] The LmEaster gene was cloned from adult locusts. Total RNA was extracted from the locusts, and cDNA was synthesized via reverse transcription. Using the cDNA as a template, and with LmEaster-F (5'-TGGCAACACATTACGCAG-3') and LmEaster-R (5'-CTCCATCATACTTGGGGTG-3') as primers, PCR was performed to amplify the LmEaster gene. The resulting LmEaster gene sequence is shown in SEQ ID NO:2.

[0053] 2. Synthesis of dsRNA

[0054] dsRNA was further synthesized based on the LmEaster gene. Primers for LmEaster RNAi and control GFP RNAi primers were designed; primer sequences are shown in Table 1. cDNA was used as a template for PCR amplification to obtain the target fragment, which was then stored at 4°C. The purified product was then used to synthesize LmEaster and GFP double-stranded RNA according to the T7 RiboMAX system (Promega, Madison, WI, USA) kit instructions. The concentrations of dsLmEaster and dsGFP were detected using a NanoPhotometer micro-spectrophotometer, and the concentrations of both solutions were adjusted to 1000 ng / mL and stored at -20°C for later use.

[0055] Table 1 dsRNA primer sequences

[0056] Primer name Primer sequence (5'-3') Product size dsLmEaster-1-F GGATCCTAATACGACTCACTATAGG TTCCCATGGATGGCACTTCT 274 bp dsLmEaster-1-R GGATCCTAATACGACTCACTATAGG CTGACCCTGACCAAGGCTAT 274 bp dsLmEaster-2-F GGATCCTAATACGACTCACTATAGG CCCACGTCCAGACAATCTTT 416 dsLmEaster-2-R GGATCCTAATACGACTCACTATAGG TATCCGAACTGACCCTGACC 416 dsLmEaster-3-F GGATCCTAATACGACTCACTATAGG GGTCAGGGTCAGTTCGGATA 355 dsLmEaster-3-R GGATCCTAATACGACTCACTATAGG TCGAAATGAAACAATGCCAA 355 dsGFP-F GGATCCTAATACGACTCACTATAGG CACAAGTTCAGCGTGTCCG 420 dsGFP-R GGATCCTAATACGACTCACTATAGG GTTCACCTTGATGCCGTTC 420 .

[0057] The dsLmEaster-1 gene sequence is shown in SEQ ID NO: 3.

[0058] 1 TTCCCATGGA TGGCACTTCT GGGATATGAA GGTAGAACCC CAGGTTCAAC TGTCTTCTCA

[0059] 61 TGTGGAGGAA CTATTATTAA CAAGAGATAC ATCTTAACAG CTGCTCACTG TGTTGCTACT

[0060] 121 GAAAACCAGA AATTGAAAC TGTTCGTTTA GGTGAGCTAA ATTTGCTAC AAATCCTGAC

[0061] 181 TGTGAGGATT ATTGTGCTGA TCCTGTGGTT GAAAGAGATG TTGAAATGAT TGTGATCCAT

[0062] 241 CCAAAGTACA ATAATCCATT TCGCAAAAAT GATA.

[0063] The dsLmEaster gene sequence is shown in SEQ ID NO: 12.

[0064] 1 CCCACGTCCA GACAATCTTT TGAGGCATAA TAATTTTCAT TTATTGAATG GCACATGTGG

[0065] 61 AGAGTTATCA GATATAAACA GAATAATATC TGGTCAGAAT GCTACTTTAG GACAATTCCC

[0066] 121 ATGGATGGCA CTTCTGGGAT ATGAAGGTAG AACCCCAGGT TCAACTGTCT TCTCATGTGG

[0067] 181 AGGAACTATT ATTAACAAGA GATACATCTT AACAGCTGCT CACTGTGTTG CTACTGAAAA

[0068] 241 CCAGAAATTG AAAACTGTTC GTTTAGGTGA GCTAAATTTG TCTACAAATC CTGACTGTGA

[0069] 301 GGATTATTGT GCTGATCCTG TGGTTGAAAG AGATGTTGAA ATGATTGTGA TCCATCCAAA

[0070] 361 GTACAATAAT CCATTTCGCA AAAATGATAT AGCCTTGGTC AGGGTCAGTT CGGATA。

[0071] The dsLmEaster gene sequence is shown in SEQ ID NO: 13

[0072] 1 GGTCAGGGTC AGTTCGGATA TCCCATATAC AGATTTTATA AGGCCAATCT GCCTTCCATT

[0073] 61 TGATAATTCA AATGACACCA AACGCCAAAT CAAATATGAG ATTGCAGGGT GGGGCAAACA

[0074] 121 GGATTCATTG GACACAAATG GAAGCACTAT ACTCCAGTTT GCAACTGTAA CTGTAACACC

[0075] 181 AATTGAGGAA TGCAGTCCTT TGCAGTCAAG GCAAGTTCAG CCATTATCAA AGGTTACACA

[0076] 241 AGTGTGTGCA GGGGGACAAG GACCTGATGC ATGCAATGGT GATAGTGGTG GTCCACTTAT

[0077] 301 GAAATACTCG AATGACTCAA GACCAGTACA ACAAGTTGGC ATTGTTTCAT TTCGA.

[0078] 3. Determination of LmEaster gene expression after RNA interference

[0079] The expression level of the LmEaster gene in locusts after interference treatment was detected. The experiment involved injecting LmEaster double-stranded RNA into three groups of locust nymphs of uniform development: an interference group and a control group. Each locust was injected with 5 mL of dsRNA solution. 24 hours after treatment, the entire locust was homogenized in a homogenizer with 500 μL of trizol added for 2 minutes. RNA was then extracted and reverse-engineered into cDNA using a reverse-engineering kit. Using pre-designed quantitative real-time PCR primers for LmEaster and the Actin gene as controls, the expression level of the LmEaster gene in each treatment group was detected using real-time quantitative PCR. The total volume of the quantitative reaction was 20 mL, including 2 mL of cDNA template, 6 mL of ddH2O, 1 mL each of forward and reverse primers (10 mmol / L), and 10 mL of 2×TB Green Premix Ex Taq (Tli RNaseH Plus). Each sample was tested in triplicate, using a 2- DDCt The relative expression level was analyzed using the method.

[0080] dsLmEasterRNA was injected into locusts, and the expression level of the LmEaster gene was detected 24 hours after each treatment. The results are as follows: Figure 1~Figure 3 As shown, when the LmEaster gene was interfered with, the expression level of the LmEaster gene in locusts was significantly reduced. Among them, the interference efficiency of dsLmEaster-1 was 91.15% ( Figure 1 The interference efficiency of dsLmEaster-2 is 70.13%. Figure 2 The interference efficiency of dsLmEaster is 83.13%. Figure 3 Therefore, dsLmEaster-1 has the highest interference efficiency and was selected for subsequent experimental arrangements.

[0081] 4. The effect of dsLmEaster on locust immune defense

[0082] Third-instar locust nymphs, pre-starved for 12 hours, were placed in sterile test baskets, with 30 nymphs per basket as one replicate. Each treatment consisted of 3 baskets (replicated 3 times), and the nymphs were fed fresh wheat seedlings until the end of the experiment. The experiment was conducted indoors at 30°C and 16L:8D.

[0083] The change in the expression level of the antimicrobial peptide gene defensin was measured 24 hours after injection of LmEaster double-stranded RNA. qPCR primers (qPCR-defensin-F: 5'-CAGCACTTCTCCTAGCCCTT-3'; qPCR-defensin-R: 5'-TTGTAGCCCTTGTTCATGGC-3') were designed, and real-time quantitative PCR was used to detect the expression level of the LmEaster gene in each treatment group. The total volume of the quantitative PCR reaction was 20 mL, including 2 mL of cDNA template, 6 mL of ddH2O, 1 mL each of forward and reverse primers (10 mmol / L), and 10 mL of 2'TB Green Premix Ex Taq (Tli RNaseH Plus). Three biological replicates were set for each sample, using a 2- DDCt Relative expression levels were analyzed using a method. Daily mortality and survival counts were recorded, and the 15-day locust survival rate was calculated.

[0084] The above experimental results indicate that, after dsLmEaster treatment alone, the expression level of LmEaster decreased, while the expression level of the locust antimicrobial peptide defensin increased. Figure 4 This indicates that the locusts' immune defense capabilities have been enhanced; and the locusts' survival rate has also increased significantly. Figure 5 ).

[0085] This invention clones the LmEaster gene and synthesizes double-stranded RNA, treats locusts by injecting dsRNA, and detects the locust survival rate between treatments, indicating that LmEaster participates in suppressing locust immune defense and plays an important role in the locust immune defense process.

[0086] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. The locust immune-related protein LmEaster gene, characterized in that, The gene encodes a protein with the amino acid sequence shown in SEQ ID NO:

1.

2. The locust immune-related protein LmEaster gene according to claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A preparation for enhancing the immunity of locusts, characterized in that, The preparation for enhancing locust immunity includes the interference sequence of the locust immune-related protein LmEaster gene as described in claim 1, wherein the nucleic acid sequence of the interference sequence of the locust immune-related protein LmEaster gene is shown in SEQ ID NO: 3.

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