Application of Locust Par-1 Gene and Its dsRNA in Locust Control

Abstract

The present invention belongs to the field of biotechnology, and relates to the prevention and treatment of migratory locusts, and in particular refers to the application of the migratory locust Partitioning defective 1 (Par‑1) gene and its dsRNA in the prevention and treatment of migratory locusts. By injecting the dsRNA of the synthesized Par‑1 gene into the body cavity of the female migratory locust, the Par‑1 gene of the migratory locust can be specifically silenced, resulting in the female migratory locusts not laying eggs and causing a large number of deaths, with a mortality rate of 100%, effectively inhibiting the reproduction and survival of migratory locusts, indicating that inhibiting the expression of the Par‑1 gene can achieve a good effect of preventing and treating migratory locusts. Due to the high efficiency, specificity and safety of the present invention, it has broad application prospects in the prevention and treatment of migratory locust pests, and has good economic benefits for protecting my country's agricultural production and ecological security.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to the prevention and treatment of migratory locusts, and particularly refers to the application of the migratory locust Partitioningdefective 1 (Par-1) gene and its dsRNA in the prevention and treatment of migratory locusts. Background Art

[0002] Pest control based on RNA interference technology has the following advantages: (1) Specificity: dsRNA only degrades mRNA homologous to it, reducing the toxic effect on non-target organisms, especially for higher animals and humans. (2) High efficiency: A small amount of dsRNA can effectively inhibit the expression of target genes, and dsRNA entering the body of pests can cause systemic and even progeny target gene silencing. (3) Safety: RNA is easily degraded in nature, leaving no residue, and does not pollute the ecological environment and crop products, making it relatively safe. At present, the basic technology for pest control based on RNA interference is to screen target sequences to obtain dsRNA with high lethality. The application of publication number CN112662690A discloses that the dsRNA of the migratory locust Rab5 gene is used to control migratory locusts. The migratory locust Rab5 gene is a key regulatory factor for cell transport; the application of a migratory locust Rab11A gene and its dsRNA in migratory locust control is disclosed. The migratory locust Rab11A gene is a migratory locust small GTPase gene.

[0003] Partitioning defective 1 (Par-1) is a cell polarity protein that plays an important role in cell polarity regulation and is one of the important protein molecules for cell polarity regulation. Cell polarity refers to the spatial asymmetric distribution of cell shape, structure and function. The main reason for this is that proteins with special functions are confined to special areas of the cell membrane. So far, there has been no report on silencing the Par-1 gene of adult locusts to kill them and achieve locust control. Summary of the invention

[0004] The present invention proposes an application of a locust Par-1 gene and a dsRNA thereof in locust prevention and control. The dsRNA prepared by using the locust Par-1 gene is injected into female locusts to prevent the locusts from laying eggs and kill them, thereby preventing locusts from breaking out and causing disasters.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The locust Par-1 gene of the present invention has an ORF sequence as shown in SEQ ID No. 1. The ORF sequence of the Par-1 gene has a full length of 2523 bp and encodes 840 amino acids.

[0007] The present invention provides a dsRNA synthesized based on the locust Par-1 gene, which is a double-stranded RNA consisting of a sense chain nucleotide shown in SEQ ID No.9 and an antisense chain nucleotide complementary to SEQ ID No.9.

[0008] The present invention also provides a primer pair for synthesizing the dsRNA, and the nucleotide sequences thereof are shown as SEQ ID No.4 and SEQ ID No.5 respectively.

[0009] The present invention also provides a method for synthesizing dsRNA based on the above locust Par-1 gene, comprising the following steps:

[0010] (1) Based on the Par-1 gene sequence shown in SEQ ID No.1, the cDNA reverse transcribed from the total RNA extracted from the ovary of the migratory locust was used as a template, and PCR amplification was performed using the upstream and downstream primers shown in SEQ ID No.4 and SEQ ID No.5 to obtain a 464 bp PCR amplification product, the nucleotide sequence of which is shown in SEQ ID No.6;

[0011] (2) The PCR amplification product was connected to the pGEM-T vector to obtain the recombinant plasmid pGEM-T-Par-1;

[0012] (3) Using the recombinant plasmid pGEM-T-Par-1 as a template, PCR amplification was performed using a primer pair with a T7 promoter sequence as shown in SEQ ID No. 7 and SEQ ID No. 8 to obtain a DNA template for synthesizing dsRNA (i.e., SEQ ID No. 6 with T7 promoters at both ends);

[0013] (4) Using DNA with T7 promoter sequence as template, use T7RiboMAX TM dsRNA was synthesized by in vitro transcription using the Express RNAi System (Promega) kit.

[0014] Application of locust Par-1 gene and / or dsRNA of locust Par-1 gene in preparing locust control agent.

[0015] Application of locust Par-1 gene and / or dsRNA of locust Par-1 gene in preparing an agent for inhibiting locust egg laying.

[0016] Furthermore, the minimum dosage of the dsRNA of the locust Par-1 gene in the above reagent is 15 μg per locust.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention can effectively inhibit the gene expression of Par-1 by injecting the synthetic dsRNA based on the Par-1 gene of the migratory locust into the female migratory locust, produce the effects of inhibiting egg laying and causing death, and the dosage of dsRNA is small (15 μg), the lethality rate can reach 100%, and the effect is remarkable. In addition, the use of dsRNA to control locusts has strong species specificity and will not cause gene interference to other species; at the same time, dsRNA is easily degraded in the natural environment and is friendly to the ecological environment. Therefore, in view of the high efficiency, specificity and safety of the present invention, it has broad application prospects in the control of migratory locust pests and has good economic benefits for protecting my country's agricultural production and ecological security.

[0019] 2. The locusts in the group injected with dsPar-1 died on the 1st or 2nd day after the injection, and all died on the 20th day after the injection, with a survival rate of 0%. Before the female locusts died, their egg-laying ability was significantly inhibited, that is, the proportion of egg-laying locusts in the group injected with dsPar-1 was only 1.2%, while 100% of the locusts in the control group laid eggs.

[0020] 3. Compared with the existing technology, the use of dsRNA as a means of controlling locusts has the advantages of specificity, high efficiency and safety. At the same time, compared with RNA interference based on other genes as targets, the advantages of interfering with the Par-1 gene for locust control are: (1) 100% lethality of adult locusts is achieved. Locusts are migratory pests that can only migrate after they have emerged as adults. When a locust plague breaks out, it is mostly the large number of migrating adults that cause damage to crops. Therefore, the control of adult locusts is an important part of locust control. At present, other genes for locust control mostly act on the molting of locust larvae to cause lethality, and their effects on adults are unknown; (2) Interference with the Par-1 gene significantly inhibits locust egg laying, ensuring that the next generation will not be produced before the locust dies, effectively controlling the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The interference efficiency of Par-1 gene in fat body tissue and ovarian tissue after double-stranded RNA injection. In the figure, **P<0.005, *P<0.01.

[0023] Figure 2 This is a statistical chart of the survival rate of locust adults after interfering with Par-1 expression.

[0024] Figure 3 This is a statistical chart of the proportion of egg-laying locusts after interfering with Par-1 expression, ****P<0.0001. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Sequence of Locust Par-1 Gene and Obtaining of dsRNA

[0027] 1. Obtaining the Par-1 gene sequence of Locust migratory locust

[0028] 1.1 Anatomy of Locust Tissue

[0029] Select healthy female adult locusts, first cut off their heads with anatomical scissors that have been sterilized with alcohol, then cut open the abdomen from the cut site, use anatomical forceps that have been sterilized with alcohol to remove the fat bodies and ovaries in the abdominal cavity and put them into 2 mL centrifuge tubes respectively, quickly place the centrifuge tubes in liquid nitrogen for instant freezing, and store them in a -80°C refrigerator for later use.

[0030] 1.2 Total RNA extraction

[0031] (1) Place the tissue obtained above on ice, add 300 μL TRIzol (Tiangen), and grind it thoroughly on ice with an electric grinding rod; let the tissue suspension stand at room temperature for 30 minutes to ensure that the tissue is fully lysed.

[0032] (2) Centrifuge at 12,000 rpm and 4°C for 5 minutes to remove most of the fat and transfer the clear lysate at the bottom to a new centrifuge tube.

[0033] (3) Add 500 μL of chloroform, shake vigorously manually for 2 minutes, place on ice for 5 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes; transfer the supernatant to a new centrifuge tube.

[0034] (4) Add 500 μL of acid phenol chloroform (phenol: chloroform: isoamyl alcohol = 25:24:1), shake vigorously manually for 2 minutes, place on ice for 5 minutes, and centrifuge at 12,000 rpm, 4°C for 10 minutes; remove the supernatant and transfer it to a new centrifuge tube.

[0035] (5) Add 500 μL of isopropanol, invert until thoroughly mixed, and incubate at -20 °C overnight.

[0036] (6) Centrifuge the RNA precipitated overnight at 12,000 rpm and 4°C for 10 minutes, discard the supernatant and retain the precipitate.

[0037] (7) Add 500 μL of pre-chilled 75% ice ethanol to the precipitate, centrifuge at 12,000 rpm, 4 °C for 10 min, and discard the supernatant.

[0038] (8) Repeat the above step (7) to obtain RNA precipitate.

[0039] (9) Place the RNA precipitate on a clean bench and blow dry it for 30 minutes to dry the RNA.

[0040] (10) Add an appropriate amount of ultrapure water to the tube containing RNA and incubate at 4°C to allow the RNA to fully dissolve.

[0041] (11) After the RNA is fully dissolved, gently shake to mix, use 1% agarose gel electrophoresis to check the integrity of the RNA, and use NanoDrop 2000 to measure the RNA concentration. Then dilute the RNA to 500 ng / μL with ultrapure water for use.

[0042] 1.3 Reverse transcription

[0043] The total RNA was reverse transcribed into the first cDNA as follows:

[0044] (1) Take 2 μg of total RNA, add water to 8 μL, incubate at 65 ˚C in a PCR instrument for 5 minutes, and quickly cool on ice.

[0045] (2) Add 2 μL of 5× gDNA Buffer, mix gently and centrifuge briefly, incubate at 42 ˚C in a PCR instrument for 3 minutes, and quickly cool on ice.

[0046] (3) Prepare the reaction system mixture as shown in Table 1. The components and amounts are shown in the following table:

[0047] Table 1 Reaction system for reverse transcription into the first cDNA

[0048]

[0049] (4) Add 10 μL of the above mixture to the system in (3), mix gently and centrifuge briefly.

[0050] (5) Incubate in a PCR machine at 42 ˚C for 15 minutes, then at 95 ˚C for 3 minutes, and then place on ice to obtain cDNA.

[0051] 1.4 Obtaining the Par-1 gene ORF sequence

[0052] Based on the sequence information of Par-1 proteins of known insects, homology comparison was used to search in the locust transcriptome to obtain the transcript of locust Par-1, and further combined with the locust genome information to splice a complete locust Par-1 gene. The upstream and downstream primer sequences (as shown in SEQ ID No. 2 and SEQ ID No. 3) were designed using primer premier5.0 software and sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for synthesis.

[0053] The upstream and downstream primer sequences for PCR amplification of Par-1 gene are as follows:

[0054] Par-1-F (SEQ ID No. 2): 5'-ATGCACACGGAAATGCCACT-3';

[0055] Par-1-R (SEQ ID No. 3): 5'-TCACAGTTTCAGTTCATTCG-3'.

[0056] The cDNA reverse transcribed in step 1.3 was used as a template, and the upstream and downstream primers shown in sequences SEQ ID No. 2 and SEQ ID No. 3 were combined to obtain the ORF fragment of the Par-1 gene by PCR amplification. This fragment was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing (all subsequent primer synthesis and sequencing were completed by Beijing Liuhe BGI Gene Technology Co., Ltd.). The ORF sequence of the Par-1 gene is shown in SEQ ID No. 1. The full length of the ORF sequence is 2523 bp and encodes 840 amino acids.

[0057] 2. Synthetic dsRNA

[0058] 2.1 DNA template for amplification of dsRNA

[0059] Based on the Par-1 gene sequence shown in SEQ ID No.1, upstream and downstream primers for synthesizing the DNA molecular template sequence of dsPar-1 (the positive chain is shown in SEQ ID No.6) were designed using primer premier5.0, and the primer sequences are shown in SEQ ID No.4 and SEQ ID No.5, respectively.

[0060] Using the cDNA reverse transcribed in step 1.3 as a template, PCR amplification was performed using the upstream and downstream primers shown in SEQ ID No. 4 and SEQ ID No. 5. The reaction system is shown in Table 2.

[0061] dsPar-1-F (SEQ ID No. 4): 5'-ATGTTGAACCTGAGCCTGACTT-3';

[0062] dsPar-1-R (SEQ ID No. 5): 5'-CGTTGCTGCACTTGCTGAAT-3'.

[0063] Table 2 Reaction system for PCR amplification using cDNA as template

[0064]

[0065] The reaction conditions were as follows: (1) 95°C, 3 min; (2) 95°C, 30 sec; 56°C, 30 sec; 72°C, 30 sec; 35 cycles; (3) 72°C extension for 5 min; and storage at 4°C.

[0066] The single product obtained by PCR amplification was connected to the pGEM-T (Tiangen) vector to obtain the recombinant plasmid pGEM-T-Par-1. Sequencing results showed that the size of the PCR amplification product was 464 bp.

[0067] Using the recombinant plasmid pGEM-T-Par-1 obtained above as a template, PCR amplification was performed with primers with T7 promoter sequence to obtain a DNA template for synthesizing dsRNA (a nucleotide sequence of T7 promoter added to both ends of a 464 bp sequence as shown in SEQ ID No. 6). The PCR reaction system and conditions were the same as those in Table 2. The target fragment was purified using the Wizard® SV Gel and PCR Clean-Up System (Promega) kit. The concentration was measured by NanoDrop 2000 and determined to be between 125 ng / μl and 1 mg / μl.

[0068] The upstream and downstream primer sequences of the DNA template for synthesizing dsRNA are the 5' end of the sequence SEQ ID No.4 and the sequence SEQ ID No.5 plus the T7 promoter sequence, as follows (the T7 promoter sequence is underlined):

[0069] dsPar-1-T7-F (SEQ ID No.7):

[0070] 5'- TAATACGACTCACTATAGG ATGTTGAACCTGAGCCTGACTT-3′;

[0071] dsPar-1-T7-R (SEQ ID No.8):

[0072] 5'- TAATACGACTCACTATAGG CGTTGCTGCACTTGCTGAAT-3'.

[0073] 2.2 Synthesis of dsRNA

[0074] Using the DNA with T7 promoter sequences at both ends obtained in step 2.1 as a template, use T7RiboMAX TM dsRNA was synthesized by in vitro transcription using the ExpressRNAi System (Promega) kit.

[0075] The in vitro transcription reaction system is shown in Table 3.

[0076] Table 3 In vitro transcription reaction system

[0077]

[0078] The reaction conditions were: 37°C, 60 minutes; 70°C, 10 minutes; 25°C, 30 minutes.

[0079] 2.3 Purification of dsRNA

[0080] (1) Add 160 μL Nuclease-Free water and 200 μL acidic phenol chloroform to the reaction system in step 2.2, mix thoroughly and place on ice for 5 minutes;

[0081] (2) Centrifuge at 13,000 g for 10 min at 4°C and transfer the supernatant into a new 1.5 mL centrifuge tube.

[0082] (3) Add 0.1 volume of sodium acetate (3.0 M, pH 5.2) and 2.5 volume of anhydrous ethanol to the supernatant, mix thoroughly, and place on ice for 5 minutes;

[0083] (4) Centrifuge at 13,000 g for 10 min at 4°C and discard the supernatant.

[0084] (5) Add 500 μL of pre-cooled 75% ethanol, centrifuge at 13,000 g for 10 min at 4°C, and discard the supernatant.

[0085] (6) Repeat step (5) and wash again with 75% ethanol;

[0086] (7) Place in a clean bench to dry for 20 minutes, add 40 μL of Nuclease-Free water, place on ice to dissolve for 20 minutes, mix well, measure the concentration using NanoDrop 2000, and place at -20˚C for later use.

[0087] The obtained dsRNA was sequenced. The sequencing results showed that the dsRNA of the locust Par-1 gene was a double-stranded RNA consisting of a sense strand and an antisense strand, the nucleotide sequence of the sense strand was shown in SEQ ID No.9, and the nucleotide sequence of the antisense strand was the reverse complementary sequence of SEQ ID No.9.

[0088] Example 2: Experiment on the lethality of locusts by synthesizing dsRNA from the locust Par-1 gene

[0089] 1. Locust dsRNA Injection

[0090] Female locusts within 12 hours after emergence were selected as experimental injection materials and randomly divided into dsPar-1 treatment group and dsGFP control group (locusts injected with dsGFP), 20 in each group, repeated 3 times. 15μg dsRNA (concentration of about 5μg / μL, about 3μL) was injected into the body from the second and third intersegments of the locust abdomen using a 10μL microinjector. The injected locusts were placed in a well-ventilated metal cage (25cm×25cm×25cm) for breeding at a temperature of 30±2℃ and a photoperiod of 14L:10D. They were fed with fresh wheat seedlings twice a day. The mortality of adults was observed and counted every day after injection, and the statistics were stopped after the first oviposition of females.

[0091] 2. Par-1 gene interference efficiency detection

[0092] 48 hours after dsRNA injection, 8 rats were randomly selected from the dsPar-1 treatment group and the dsGFP control group, and the fat body and ovary tissues were dissected to detect the interference efficiency of Par-1.

[0093] The cDNA of fat body and ovary tissues were obtained according to the method of Example 1. Then, according to the instructions of Tiangen SuperReal Fluorescence Quantitative Premix Reagent Enhanced Edition (SYBR Green) Kit (PF205), Par-1 upstream and downstream primers as shown in SEQ ID No.10 and SEQ ID No.11 and β-actin upstream and downstream primers as shown in SEQ ID No.12 and SEQ ID No.13 were used to perform qRT-PCR reaction on LightCycler 96 (Roche) to detect the relative expression levels of the target gene (Par-1) and the internal reference gene (β-actin), and then according to 2 -ΔΔCt The results are shown in Table 1. Figure 1 As shown, compared with the dsGFP control group, the expression of Par-1 in the dsPar-1 treated group decreased by 71% and 46% in the fat body and ovary, respectively, indicating that Par-1 was effectively silenced.

[0094] The primer sequences for qRT-PCR reaction are as follows:

[0095] Par-1-qRT-F (SEQ ID No. 10): 5'- CGGTGGAGGAGTGACAATGGT-3';

[0096] Par-1-qRT-R (SEQ ID No. 11): 5'-ATTGCGAGGGAAGGGCTGTC-3'.

[0097] β-actin-qRT-F (SEQ ID No. 12): 5'-AATTACCATTGGTAACGAGCGATT-3';

[0098] β-actin-qRT-R (SEQ ID No. 13): 5'-TGCTTCCATACCCAGGAATGA-3'.

[0099] The qRT-PCR reaction system is shown in Table 4.

[0100] Table 4 qRT-PCR reaction system

[0101]

[0102] The reaction conditions were as follows: (1) pre-denaturation at 95 °C for 10 min; (2) 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s; (3) melting curve analysis, 95 °C for 60 s, 65 °C for 30 s, and 95 °C for 30 s.

[0103] 3. Statistics on mortality and egg-laying ratio of migratory locusts after dsRNA injection

[0104] The control group injected with dsGFP laid eggs within 7-9 days after injection, with a survival rate of 94%. There was no significant difference in egg-laying behavior, egg-laying rate and vital signs compared with normally developed female adults, and the proportion of egg-laying locusts was 100%.

[0105] Table 5 Statistical results of the survival rate of migratory locusts after injection of dsRNA

[0106]

[0107] The survival rate statistics are shown in Table 5 and Figure 2 As shown by Figure 2 As shown in Table 5, the dsPar-1 injected group died on the 1st and 2nd day after injection, and all died on the 20th day after injection, except for one locust that died after laying eggs. That is, the proportion of locusts that laid eggs in the dsPar-1 injected group was 1.9%, and the survival rate was 0%. Figure 3As shown in Figure 3, it can be seen that the proportion of locusts laying eggs in the treatment group injected with dsPar-1 was only 1.2%, while 100% of the locusts in the control group laid eggs.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A dsRNA of the Locust Par-1 gene, characterized in that: The sequence of the dsRNA is shown as SEQ ID No.

9.

2. The method for preparing the dsRNA of the locust Par-1 gene according to claim 1, characterized in that: The steps are: (1) Using the cDNA of the locust ovary as a template, PCR amplification was performed using primers dsPar-1-F and dsPar-1-R to obtain an amplified product; (2) Insert the amplified product into the pGEM-T vector to obtain the recombinant plasmid pGEM-T-Par-1; (3) Using the recombinant plasmid pGEM-T-Par-1 as a template, PCR amplification and restriction digestion were performed with primers dsPar-1-T7-F and dsPar-1-T7-R to obtain the target fragment; (4) Using the target fragment as a template, dsRNA is synthesized by in vitro transcription.

3. The method for preparing the dsRNA of the locust Par-1 gene according to claim 2, characterized in that: The nucleotide sequence of the primer dsPar-1-F in the step (1) is shown in SEQ ID No.4, the nucleotide sequence of the primer dsPar-1-R is shown in SEQ ID No.5, and the nucleotide sequence of the amplified product is shown in SEQ ID No.

6.

4. The method for preparing the dsRNA of the locust Par-1 gene according to claim 3, characterized in that: The nucleotide sequence of the primer dsPar-1-T7-F in step (3) is shown in SEQ ID No. 7, and the nucleotide sequence of the primer dsPar-1-T7-R is shown in SEQ ID No.

8. The nucleotide sequence of the target fragment is the sequence shown in SEQ ID No. 6 with T7 promoters at both ends.

5. The method for preparing the dsRNA of the locust Par-1 gene according to claim 4, characterized in that: The in vitro transcription is achieved using an in vitro transcription kit.

6. Use of the dsRNA of the locust Par-1 gene according to claim 1 in preparing a locust control agent.

7. Use of the dsRNA of the locust Par-1 gene according to claim 1 in preparing an agent for inhibiting locust egg laying.

8. The use according to claim 6 or 7, characterized in that: The ORF sequence of the locust Par-1 gene is shown as SEQ ID No.

1.

9. The use according to claim 6 or 7, characterized in that: The minimum dosage of the dsRNA of the locust Par-1 gene in the reagent is 15 μg per locust.

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