Lmserpinb1 gene and use thereof

Abstract

The application relates to the field of agricultural biotechnology, in particular to an LMSerpinb1 gene and application thereof. A locusta migratoria serpinb1 gene is cloned, and the locusta migratoria mortality and the gene expression amount change of the target gene in the locusta migratoria are detected through the method of mixed feeding of the locusta migratoria with metarhizium anisopliae, so that the role of the locusta migratoria in the metarhizium anisopliae infection is verified, and a foundation for further clarifying the action mechanism of Serpin is laid.

Description

Technical Field

[0001] This application relates to the field of agricultural biotechnology, specifically to the LMSerpinb1 gene and its applications. Background Technology

[0002] Serine protease inhibitors (Serpins) are a large and widely distributed family of protease inhibitors. Studies have found that Serpins are present in all organisms and participate in regulating various physiological responses in insects, such as immune melanization and innate immune responses like tissue formation during growth and development. Serpins participate in the Toll pathway and melanization response by regulating the activity of serine proteases in the body. They primarily utilize a suicide substrate principle to specifically form stable covalent complexes with target enzymes, leading to enzyme inactivation and terminating the insect's excessive immune response, thus preventing damage to the insect itself. Summary of the Invention

[0003] The purpose of this invention is to provide the LMSerpinb1 gene.

[0004] Another object of the present invention is to provide the application of the LMSerpinb1 gene.

[0005] The LMSerpinb1 gene according to the present invention encodes a protein with an amino acid sequence as shown in SEQ ID NO: 1.

[0006] SEQ ID NO: 1

[0007]

[0008]

[0009] The LMSerpinb1 gene according to the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0010] SEQ ID NO: 2:

[0011]

[0012] This application provides the application of the above-mentioned LMSerpinb1 gene for locust control.

[0013] The method for controlling locusts according to this application includes the following steps:

[0014] Locusts were infected with Metarhizium anisopliae, and simultaneously fed with an interference fragment of the LMSerpinb1 gene of this invention. The interference fragment was obtained by amplifying the total cDNA of the locusts using the following primers.

[0015] B2-F:5'- TAATACGACTCACTATAGGTAGCCAATCAGAAACAGCAAGG-3”

[0016] B2-R:5'- TAATACGACTCACTATAGG TCTCGTAATACCAGTGAAGTTCGC-3';

[0017] C2-F:5'- TAATACGACTCACTATAGG CGATGCTACAATGCGTGA-3',

[0018] C2-R:5'- TAATACGACTCACTATAGG TTCAAGGTTGCTCAAGCC-3'.

[0019] This experiment cloned the locust serpinb1 gene and, by feeding locusts with Metarhizium anisopliae, detected changes in locust mortality and gene expression levels of the target gene, verifying its role in locust infection by Metarhizium anisopliae and laying the foundation for further clarifying the mechanism of action of Serpin. Attached Figure Description

[0020] Figure 1 Displaying gene clone gel images;

[0021] Figure 2 Showing the relative gene expression levels at different ages;

[0022] Figure 3 Showing the relative gene expression levels in different tissues;

[0023] Figure 4 Showing the relative gene expression levels after infection with Metarhizium anisopliae;

[0024] Figure 5 Showing the relative expression levels of genes with different interfering fragments;

[0025] Figure 6 The survival rates of the Oriental migratory locust under different treatments are shown;

[0026] Figure 7A This shows the changes in the expression level of the Defension gene in locusts after different treatments;

[0027] Figure 7B This shows the changes in the expression level of the sepinB1 gene in locusts after different treatments;

[0028] Figure 7C Showing changes in PPAE gene expression levels in locusts after different treatments;

[0029] Figure 7D This shows the changes in PPO gene expression levels in locusts after different treatments. Detailed Implementation

[0030] Experimental materials: The test Metarhizium strain IMI330189, Locusta migratoria manilensis eggs were collected in Cangzhou, Hebei Province, and hatched to the 3rd instar in an artificial climate incubator.

[0031] Example 1 Gene cloning

[0032] The locust adults were placed in liquid nitrogen and then ground into powder in a mortar. The total RNA of the locust was extracted by the Trizol reagent method solution, and reverse transcription was performed by using the 5x ALL-IN-One RT MasterMix reagent kit to synthesize cDNA. The cDNA was used as a template, and the primer sequences were F: 5' ATGACAGCGACCATTCAGTAC 3' and R: 5' TGGCGGTGAGGAGTAGC 3' to perform PCR amplification of the serpinb1 gene. The PCR reaction conditions were as follows: 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, a total of 35 cycles, 72°C extension for 10 min, and finally 4°C storage. The PCR product was separated and purified by 1% agarose gel electrophoresis. The purified product was connected to the pMD19-T Vector, transformed into E. coli competent cells DH5α by heat shock, and positive clones were screened by ampicillin resistance. PCR verification was performed, and sequencing was performed by Shanghai Shenguo Bio (Beijing) Co., Ltd.

[0033] As shown in Figure 1 , the RNA of the locust midgut was extracted and reverse transcribed to obtain cDNA, and the serpinb1 gene sequence was obtained by PCR amplification. The analysis showed that the full-length sequence was 1260 bp, which encoded 395 amino acid residues, the encoded protein was 48.6 KD, and the isoelectric point was 5.23.

[0034] Example 2 Determination of the relative expression amount of the target gene in different tissues and different instars of locusts

[0035] The bodies of the first to fifth instar locusts and male and female adults of Locusta migratoria manilensis were extracted, and the body wall, testis and ovary, fat body, hemolymph and hind leg, midgut and other tissues of the adults were extracted. Each was placed in liquid nitrogen, and the total RNA was extracted by the TRIzol method and reverse transcribed into cDNA. Each three tissues were a repeat, and there were three repeats. The gene expression amount of the target gene was detected by RT-qpcr method.

[0036] As shown in Figure 2 , the expression amount of the target gene was the highest at the second instar, as shown in Figure 3 , among the various tissues, the gene expression amount of the hind leg was the highest, followed by the fat body and the epidermis.

[0037] Example 3 Effect of Metarhizium infection on gene expression amount

[0038] Bait preparation: Fresh Metarhizium spore powder was added to 0.1% Tween test tube, mixed evenly, and the spore number was recorded under a microscope to make a spore suspension with a final concentration of 2.3 x 10 8 spores / mL. 1 mL of the spore suspension was mixed with 1 g of sterilized wheat bran containing 5% plant oil to make bait.

[0039] Experimental method: First, the third instar Locusta migratoria manilensis nymphs were starved for 12 h, fed with prepared bait for 24 h, and then sampled at 24 h and 48 h after Metarhizium infection. The expression level of the gene was determined by QPCR.

[0040] As shown in Figure 4 , the expression level of the target gene decreased significantly at 24 h and 48 h after Metarhizium treatment, indicating that the expression level of the target gene changed when the fungus invaded the body of Locusta migratoria manilensis. Therefore, it can be inferred that the target gene may be involved in the immune response of Locusta migratoria manilensis when the fungus invades.

[0041] Example 4 Comparison of interference efficiency of different interference fragments

[0042] The LMSerpinb1 gene was amplified with the following primers to obtain interference fragments A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, and F1.

[0043] A1-F: 5’- TAATACGACTCACTATAGG ACTTGAGGATTCTGGCTGC-3’,

[0044] A1-R: 5’- TAATACGACTCACTATAGG CGATGCCTTTCTCTATTGC-3’;

[0045] A2-F: 5’- TAATACGACTCACTATAGG ACTTGAGGATTCTGGCTGC-3’,

[0046] A2-R: 5’- TAATACGACTCACTATAGG CTTGCTGTTTCTGATTGGCT-3’;

[0047] B1-F: 5’- TAATACGACTCACTATAGG TAGCCAATCAGAAACAGCAAGG-3’,

[0048] B1-R: 5’- TAATACGACTCACTATAGG CCTTGTATGGAAGTGCGAG-3’;

[0049] B2-F: 5’- TAATACGACTCACTATAGG TAGCCAATCAGAAACAGCAAGG-3’,

[0050] B2-R: 5'- TAATACGACTCACTATAGG TCTCGTAATACCAGTGAAGTTCGC-3';

[0051] C1-F: 5'- TAATACGACTCACTATAGG CGATGCTACAATGCGTGA-3',

[0052] C1-R: 5'- TAATACGACTCACTATAGG CCAGAACTCCAGAAGGGAT-3';

[0053] C2-F: 5'- TAATACGACTCACTATAGG CGATGCTACAATGCGTGA-3',

[0054] C2-R: 5'- TAATACGACTCACTATAGG TTCAAGGTTGCTCAAGCC-3';

[0055] D1-F: 5'- TAATACGACTCACTATAGG TCCCTTCTGGAGTTCTGGAC-3',

[0056] D1-R: 5'- TAATACGACTCACTATAGG AAGTTCGCCTTGGTCTTGC-3';

[0057] D2-F: 5'- TAATACGACTCACTATAGG TCCCTTCTGGAGTTCTGGAC-3',

[0058] D2-R: 5'- TAATACGACTCACTATAGG CCATTTCCTCGTGTAGGTAGG-3';

[0059] E1-F: 5'- TAATACGACTCACTATAGG TCGCAGATTCTCGCACTT-3',

[0060] E1-R: 5'- TAATACGACTCACTATAGG CCTCAACGAAAGCCTTGTG-3';

[0061] E2-F: 5'- TAATACGACTCACTATAGG TCGCAGATTCTCGCACTT-3',

[0062] E2-R: 5'- TAATACGACTCACTATAGG ATGAGGAACAGGAACGGGT-3';

[0063] F1-F: 5'- TAATACGACTCACTATAGG ACCAAGGCGAACTTCACTG-3'

[0064] F1-R:5'- Figure 5 ATGAGGAACAGGAACGGGT-3'.

[0065] As TAATACGACTCACTATAGG shown, except for fragment F1, the rest of the fragments have interference effect, but the interference efficiency of B2 and C2 fragments is the highest, so one of them can be selected, and B2 fragment is selected in the subsequent experiment.

[0066] Example 5 Verification of gene function by gene silencing

[0067] Experimental materials: five instar locusts, Metarhizium anisopliae 189, reagent kit (T7 RiboMAXTM Express RNAi System interference kit).

[0068] Experimental method: first, use the kit to synthesize dsSerpinA1 and dsGFP, and make their final concentration 1 μg / μl. The interference sequence is

[0069] GFP-F:5'- TAATACGACTCACTATAGG ATGAGTAAAGGAGAAGAAC-3'

[0070] GFP-R:5'- TAATACGACTCACTATAGG CTATTTGTATAGTTCATCC-3'

[0071] B2-F:5'- TAATACGACTCACTATAGG TAGCCAATCAGAAACAGCAAGG-3'

[0072] B2-R:5'- Treatment TCTCGTAATACCAGTGAAGTTCGC-3'

[0073] Four treatments: dsGFP, dsRNA, Ma (Metarhizium anisopliae), Ma (Metarhizium anisopliae) + dsRNA, 15 locusts in each treatment, four replicates, dsGFP as control.

[0074] First, starve the locusts for 12 h, inject 5 uL dsRNA, and put them into wheat bran, mix Metarhizium anisopliae into the wheat bran (the number of Metarhizium anisopliae spores in each treatment is 2.5×10 8 ), and feed them with fresh wheat seedlings after 24 h of feeding with the mixed wheat bran. The specific treatment can be seen in Table 1 below.

[0075] Wheat bran configuration: add fresh Metarhizium anisopliae spore powder to 0.1% Tween test tube, mix evenly, and record the number of spores under a microscope, and prepare the final concentration of 2.3×10 8A spore suspension of 1 x 106 spores / mL. 1 mL of spore suspension was mixed with 1 g of sterilized wheat bran containing 5% plant oil to make the bait.

[0076] Table 1

[0077] Metarhizium content (spores / g) Content (μg / μl) dsGFP dsRNA 0 5 Ma (Metarhizium) 0 5 Ma (Metarhizium) + dsRNA 2.5 x 10 8 ]] 0 Figure 6 2.5 x 10 8 ]]> 5

[0078] As Figure 7A shown by the survival rate, on the ninth day of observation, the survival rate of the control group and the group with gene interference alone was the same, the survival rate of the group infected with Metarhizium alone was reduced to 40%, and the survival rate of the group treated with Metarhizium infection and gene interference was reduced to 10%, which was significantly different from the survival rate of the group infected alone. It can be seen that interfering with this gene alone has no effect on the survival of Locusta migratoria manilensis, but when infected with fungi, it can promote fungal infection, thereby increasing the insecticidal rate of Metarhizium, and it is inferred that this gene plays a certain role in the immune process of locusts.

[0079] Example 6 Expression of Immune-related Genes

[0080] In the natural immune process of insects, it is divided into humoral immunity and cellular immunity. Humoral immunity mainly includes two mechanisms of melanin formation and antibacterial peptide production, among which antibacterial peptides play a role in killing bacteria. Melanin is an important component of the hard exoskeleton of insects, which plays a barrier role. The experimental method is the same as that of Example 5, and the whole head is sampled, and the gene expression is determined by Qpcr. The Qpcr primer sequence is:

[0081] SerpinB1-F: 5' CCATCTACCTGCTAACGACG 3',

[0082] SerpinB1-R: 5' TCCTTGCTGTTTCTGATTGG 3';

[0083] PPO-F: 5' AAAGACCGCAGAGGAGAA 3',

[0084] PPO-R: 5' CCAACGATAGAACACAGGA 3';

[0085] PPAE-F: 5' CACCAGCACAAATGAATGAC 3',

[0086] PPAE-R: 5' CAACGACAATGAGGCACAG 3';

[0087] Defensin-F: 5' CCAGAAAGCGATGATGCCACTA 3',

[0088] Defensin-R: 5' CACCACAAATCAACGCCAAAGT 3'.

[0089] As Figure 7B The change of the expression of the antibacterial peptide gene under different treatment conditions was determined. As shown in the figure, the antibacterial peptide will decrease in the two treatments of Metarhizium and Metarhizium gene interference, which shows that the fungus and the interference of this gene play the same role in the immune process of locusts.

[0090] As Figure 7C As shown, the expression of the target gene will decrease when Metarhizium is interfered, which has the same effect as interference, so it can be explained that completely interfering with the gene will completely lose the effect of the gene in the immune process of locusts, thus reducing the immune ability of locusts and further reducing the survival rate, and promoting the insecticidal effect of Metarhizium.

[0091] As Figure 7D and ​ As shown, PPAE and PPO are related genes in the melanization reaction, which positively regulate the melanization reaction. As shown in the figure, the expression of the two genes will decrease in the three treatments, thus reducing the immune process of the melanization reaction of locusts, thereby increasing the mortality rate of locusts.

[0092] The above examples are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.

Claims

1. Interference sequence of locust LMSerpinbl gene, characterized in that, The locust LMSerpinb1 gene encodes a protein with an amino acid sequence as shown in SEQ ID NO: 1, and the interference sequence of the locust LMSerpinb1 gene is obtained by amplifying the total cDNA of the locust with the following primers, B2-F:5 ， - TAATACGACTCACTATAGG TAGCCAATCAGAAACAGCAAGG-3 ， , B2-R:5 ， - TAATACGACTCACTATAGG TCTCGTAATACCAGTGAAGTTCGC-3 ， ; or C2-F:5 ， - TAATACGACTCACTATAGG CGATGCTACAATGCGTGA-3 ， , C2-R:5 ， - TAATACGACTCACTATAGG TTCAAGGTTGCTCAAGCC-3 ， .

2. The interference sequence of the locust LMSerpinb1 gene according to claim 1 is used for increasing the insecticidal rate of Metarhizium on locusts.

3. A method of controlling locusts, characterized by, The method comprises the following steps: infesting the locusts with Metarhizium while interfering with the expression of the locust LMSerpinb1 gene, which encodes a protein with an amino acid sequence as shown in SEQ ID NO: 1, wherein the interference fragment for interfering with the locust LMSerpinb1 gene is obtained by amplifying the total cDNA of the locust with the following primers, B2-F:5 ， - TAATACGACTCACTATAGG TAGCCAATCAGAAACAGCAAGG-3 ， , B2-R:5 ， - TAATACGACTCACTATAGG TCTCGTAATACCAGTGAAGTTCGC-3 ， ; or C2-F:5 ， - TAATACGACTCACTATAGG CGATGCTACAATGCGTGA-3 ， , C2-R:5 ， - TAATACGACTCACTATAGG TTCAAGGTTGCTCAAGCC-3 ， .

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