A siRNA targeting the HDAC3 gene of cotton bollworm and its application in pest control

Abstract

The present invention discloses a siRNA targeting the HDAC3 gene of cotton bollworm, the nucleotide sequence of the sense chain is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.14. Also disclosed is the application of the aforementioned siRNA in the regulation of cotton bollworm growth, development or metamorphosis, the application in inhibiting the expression of the HDAC3 gene of cotton bollworm, or the application in preventing and controlling cotton bollworm, or the application in preparing cotton bollworm insecticide. Compared with dsRNAs, selecting siRNA to silence the target gene of cotton bollworm is a more accurate and effective method. The present invention finds one of the five siRNAs with outstanding silencing efficiency on the HDAC3 gene, and has a significant inhibitory effect on the pupation, eclosion, egg laying and hatching of cotton bollworm, and has an outstanding interference effect. It is expected to be applied to the prevention and control of cotton bollworm in the field, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an siRNA targeting an HDAC3 gene of cotton bollworm and an application thereof in pest control. Background Art

[0002] Helicoverpa armigera (Hübner), Lepidoptera, Noctuidae, is widely distributed in China and around the world. It is an omnivorous agricultural pest with more than 300 host species. It mainly harms crops such as cotton, corn, wheat, barley, tomatoes, green peppers, and melons, causing huge losses to the agricultural economy. Chemical pesticides and Bt insect-resistant cotton have appeared on the market, but they are prone to pesticide pollution and bollworm resistance. Therefore, it is particularly important to develop new pest control methods.

[0003] Histone deacetylase (HDAC) is a class of proteases that play an important role in the structural modification of chromosomes and the regulation of gene expression. HDAC binds tightly to negatively charged DNA, making chromatin densely curled, thereby inhibiting gene transcription. In the cell nucleus, histone acetylation and histone deacetylation are in dynamic equilibrium and are co-regulated by histone acetyltransferase and histone deacetylase. HDAC3 belongs to class I HDAC, which is ubiquitous and located in the cell nucleus. HDAC3 affects and participates in the expression of protein-coding genes for insect growth, development and metamorphosis regulation by affecting the acetylation level of histones. In Drosophila S2 cells, RNAi-mediated silencing of HDAC3 can lead to cell growth inhibition and disorders of sox14, and molting genes eip74ef, nvy, etc.; RNAi-mediated knockout of the HDAC3 gene in the larvae of Tribolium castaneum affects its development, resulting in abnormal folding of the wings of pupae and adults.

[0004] siRNA (Small interfering RNA) is a double-stranded RNA of 20 to 25 nucleotides in length. It achieves restricted selection of targets and directly targets specific genes through complementary base pairing, resulting in gene silencing phenomenon of efficient and specific degradation of mRNA. siRNA is formed by dsRNA cut by RNaseIII (such as Dicer) in cells. dsRNA can be exogenous, such as viral RNA replication intermediates or artificially introduced dsRNA; it can also be endogenous, such as dsRNA formed by single-stranded RNA in cells under the action of RNA-dependent RNA polymerase. There is a natural RNA interference (RNAi) phenomenon in plants. Fire et al. first discovered the effective and specific genetic interference of exogenous double-stranded RNA (dsRNA) on Caenorhabditis elegans. By introducing dsRNA to inhibit the expression of endogenous genes in insects, the function of the gene can be studied based on the phenotype of gene deletion. This method has been mastered by insect researchers, and the functions of various insect genes have gradually been determined. Injecting dsRNA into insects is one of the most widely used pest control methods. However, FU J (Resistance to RNA interference by plant-derived double-strandedRNAs but not plant-derived short interfering RNAs in Helicoverpa armigera[J / OL].Plant Cell and Environment, 2022, 45(6):1930-1941. DOI:10.1111 / pce.14314.) et al. found that the activity of dsRNase in the intestinal fluid of cotton bollworm is high, and the ingested dsRNA is rapidly degraded in the intestinal fluid. In contrast, siRNA is relatively stable in the digestive system, which is also the main reason for the low efficiency of dsRNA silencing target genes in cotton bollworm. These results indicate that plant-derived siRNAs may be more effective than dsRNAs in triggering the RNAi pathway of lepidopteran insects, which will help optimize plant-mediated RNAi strategies for pest control. Summary of the invention

[0005] The object of the present invention is to provide a siRNA targeting the HDAC3 gene of cotton bollworm in order to solve the above problems. The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.14.

[0006] Another object of the present invention is to provide the use of the above siRNA in regulating the growth, development or metamorphosis of cotton bollworm.

[0007] In the above-mentioned application technology scheme, the siRNA inhibits the normal pupation or emergence of cotton bollworm, reduces the normal pupation rate or emergence rate, causes the cotton bollworm to be unable to pupate normally or the pupation time is delayed, or causes the cotton bollworm to have abnormal wings and be unable to fly normally after developing into an adult.

[0008] In the above-mentioned application technology solution, the siRNA inhibits the egg laying or hatching of cotton bollworm, resulting in a significant decrease in the egg laying rate or egg hatching rate of cotton bollworm.

[0009] Another object of the present invention is to provide the use of the above siRNA in inhibiting the expression of HDAC3 gene of cotton bollworm, or in preventing and controlling cotton bollworm, or in preparing cotton bollworm insecticide.

[0010] In the above-mentioned application technology scheme, the application is to introduce the above-mentioned siRNA into cotton bollworm, thereby inhibiting the normal pupation, emergence, egg laying or hatching of cotton bollworm, thereby achieving the prevention and control of cotton bollworm.

[0011] In the above application technology scheme, the introduction is to prepare the siRNA into a solution and then introduce it into the body of the cotton bollworm, preferably by injection into the intestine of the cotton bollworm, and preferably the cotton bollworm is a larva.

[0012] Preferably, the siRNA solution is injected into the abdominal intersegmental membrane between the 6th and 7th segments of the cotton bollworm.

[0013] Preferably, the cotton bollworm is a 6th instar larva, the injection concentration of the siRNA solution is 2-4 ug / uL, and the injection volume is 2-4 uL.

[0014] In the above-mentioned application technical solution, the injection concentration of the siRNA solution is 3ug / uL, and the injection volume is 3uL.

[0015] The beneficial effects of the present invention are as follows: compared with dsRNAs, selecting siRNA to silence the target gene of cotton bollworm is a more accurate and effective method, and siRNA has the advantages of specificity, high efficiency, high stability, etc. The present invention finds one of the five siRNAs with outstanding silencing efficiency on the HDAC3 gene, and has significant inhibitory effects on pupation, eclosion, egg laying and hatching of cotton bollworm, and has outstanding interference effects, and is expected to be applied to the prevention and control of cotton bollworm in the field, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an analysis diagram of the expression pattern of HDAC3 gene in different temporal and spatial locations of cotton bollworm.

[0017] Figure 2 The results of silencing efficiency test of five siRNAs 12 hours after injection.

[0018] Figure 3 This is a graph analyzing the silencing efficiency at different times after injection of siHaHDAC3.

[0019] Figure 4 This is a comparison of the pupation status of cotton bollworm after injection of siHaHDAC3.

[0020] Figure 5 This is a comparison of the emergence of cotton bollworms after injection of siHaHDAC3.

[0021] Figure 6 This is a comparison of the egg-laying situation of cotton bollworm after injection of siHaHDAC3.

[0022] Figure 7 This is a comparison of the hatching conditions of cotton bollworms after injection of siHaHDAC3. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological reagents used are all conventional reagents in the art unless otherwise specified.

[0025] Example 1

[0026] 1. Amplification primer design

[0027] The cDNA sequence of the HDAC3 gene (HaHDAC3) of cotton bollworm was obtained using online programs such as NCBI Blast. The NCBI accession number is KM983338.1.

[0028] According to the found sequences, primer design software was used to design primers for amplification of the HDAC3 gene of cotton bollworm, and primers for amplification of an internal reference gene (RPS15, GenBank: XM-021326200.1) were designed. The primer sequences are shown in Table 1.

[0029] Table 1

[0030]

[0031] 2. Test insects

[0032] The cotton bollworm was raised indoors in the laboratory of Zhengzhou University with artificial feed. The temperature was 26±1°C, the relative humidity was 60±10%, and the photoperiod was 16 h light and 8 h dark.

[0033] 3. Synthesis of first-strand cDNA

[0034] Extract total RNA from cotton bollworm using a kit Total RNA was extracted from eggs (EG), larvae from 1st to 6th instars (L1-L6), prepupae (PP), pupae (P), female adults (FA), male adults (MA), and heads (H), integuments (I), intestines (H), and Malpighian tubules (T) of cotton bollworm from 3rd to 6th instars (Vazyme, Nanjing, China). The first-strand cDNA was synthesized using the extracted total RNA using III 1st Strand cDNA Synthesis Kit (+Gdna wiper) (Vazyme, Nanjing, China).

[0035] 4. Amplification of the HDAC3 gene of cotton bollworm

[0036] The cDNA obtained above was used as a template, and RT-qPCR reactions were performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and gene-specific primers (Table 1). RPS15 was used as an internal reference gene to correct the differences in sample cDNA results. The PCR reaction system totaled 20ul: 0.4ul of each upstream and downstream primer at a concentration of 10pmol / ul, 2ul of cDNA template, 10ul of 2×ChamQ Universal SYBR qPCR Master Mix, and the remainder was ddH 2 O. The qPCR reaction procedure is as follows:

[0037]

[0038] The results are as follows Figure 1 As shown, L1 instar, L2 instar, L3 instar, L4 instar, L5 instar, L6 instar, egg (EG), prepupa (PP), pupa (P), female adult (FA), male adult (MA), head (3H), integument (3I), midgut (3H), Malpighian tubules (3T) of L3 instar larvae, head (4H), integument (4I), midgut (4H), Malpighian tubules (4T) of L4 instar larvae, head (5H), integument (5I), midgut (5H), Malpighian tubules (5T) of L5 instar larvae, head (6H), integument (6I), midgut (6H), Malpighian tubules (6T) of L6 instar larvae. The expression levels of HDAC3 gene in the above tissues are shown. Figure 1As shown, the results showed that the HDAC3 gene was expressed in all instars and tissues of cotton bollworm, among which the expression level of HDAC3 gene in the 6th instar larvae and their tissues was relatively the highest, so the 6th instar cotton bollworm was selected as the instar for injection of siHaHDAC3.

[0039] 5. Synthesize siRNA

[0040] Through nucleotide sequence BLAST comparison in genome databases such as NCBI and ensembl, we searched for other sequences of the HDAC family of cotton bollworm, including HDAC6, HDAC7, HDAC8, and HDAC11, compared these sequences with HDAC3, selected the nucleotide sequence of the unique domain of the HDAC3 gene, designed five siRNA sequences, and commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize them. The five siRNA sequences were named: siRNA-1, siRNA-2, siRNA-3, siRNA-4, and siRNA-5.

[0041] The siRNA sequences are:

[0042] siRNA-1: positive strand: 5'-AAGGAACCACCAGUUGAGAAC-3' (SEQ ID NO. 5),

[0043] Antisense strand: 5'-GUUCUCAACUGGUGGUUCCUU-3' (SEQ ID NO. 6).

[0044] siRNA-2: positive strand: 5'-AAGAACGAAUUCUAUGAAGAC-3' (SEQ ID NO. 7),

[0045] Antisense strand: 5'-GUCUUCAUAGAAUUCGUUCUU-3' (SEQ ID NO. 8).

[0046] siRNA-3: positive strand: 5'-AACUGGUCCGGAGGUCUACAU-3' (SEQ ID NO.9),

[0047] Antisense strand: 5'-AUGUAGACCUCCGGACCAGUU-3' (SEQ ID NO. 10).

[0048] siRNA-4: positive strand: 5'-AACAUGUGUACGACAACCUCA-3' (SEQ ID NO.11),

[0049] Antisense strand: 5'-UGAGGUUGUCGUACACAUGUU-3' (SEQ ID NO. 12).

[0050] siRNA-5: sense strand: 5'-AAACCAUAUAGAGCCAGUGCU-3' (SEQ ID NO. 13), antisense strand: 5'-AGCACUGGCUCUAUAUGGUUU-3' (SEQ ID NO. 14).

[0051] 6. RNA interference

[0052] The five synthesized siRNAs were diluted with DEPC water to a final concentration of 3ug / ul as the treatment group, and the control group was injected with DEPC water.

[0053] Six-instar cotton bollworms of uniform size and consistent health were selected, and microinjection was performed on the intersegmental membrane between the 6th and 7th segments of the cotton bollworms. The injection volume of each cotton bollworm was 3ul, and the injection concentration was 3ug / ul. 100 cotton bollworms were injected into the treatment group and the control group, respectively. After the injection, the cotton bollworm larvae were placed in an artificial feed box for cultivation, and the breeding temperature was 26±1℃, the relative humidity was 60±10%, and the light cycle was: 16h light, 8h dark.

[0054] Samples from the treatment group and the control group were taken 12h, 24h, 36h, and 48h after the injection to detect the silencing efficiency of the HDAC3 gene, and RPS15 was used as an internal reference gene to correct the difference in the sample cDNA results. The detection method was RT-qPCR, and the primers, PCR reaction system, and amplification procedure were the same as those in the previous RT-qPCR method for amplifying the HDAC3 gene of cotton bollworm.

[0055] The results of the silencing efficiency test of the five siRNAs 12 hours after injection are as follows Figure 2 As shown, the silencing efficiency of siRNA-4 and siRNA-5 is good, especially the silencing efficiency of siRNA-5. siRNA-5 was selected for the subsequent injection experiment.

[0056] The silencing efficiency reached more than 90% 12 hours after the injection of siRNA-5, and the silencing efficiency could be maintained within 48 hours. Figure 3 As shown, compared with the control group, the silencing efficiencies at 12h, 24h, 36h, and 48h were 99.6%, 99.1%, 99.4%, and 94.4%, respectively.

[0057] After the injection of siRNA-5, the growth, pupation, emergence, egg laying and hatching of cotton bollworm were observed: the cotton bollworms in the siRNA-5 treatment group could not pupate normally or the pupation time was delayed, while the cotton bollworms in the CK group could pupate normally, such as Figure 4As shown in the figure, the abnormal pupation rate reached 21%. After emergence, the cotton bollworms in the treatment group developed into adults with abnormal wings and could not fly normally. The wing abnormality rate reached 28%, while the control group developed into adults with normal wings. Figure 5 After laying eggs, the number of eggs laid was counted. The number of eggs laid by cotton bollworms in the treatment group was 39% of that in the control group. Figure 6 As shown in the figure, the hatching rate of eggs was 6% in the treatment group and 82% in the control group. Figure 7 As shown. It can be seen that the HDAC3 gene plays an important role in the pupation, emergence, egg laying and hatching processes of cotton bollworm.

[0058] Based on the above experimental results, injecting siRNA that inhibits genes related to the growth and development of cotton bollworm into the insect intestine can inhibit the expression of the target gene and affect the growth and development of the pest, thereby achieving the purpose of pest control. The interference effect of siRNA-5 of the present invention is particularly significant, and it is expected to be applied to the control of cotton bollworm in the field, and has a good application prospect. Sequence Listing <110> Zhengzhou University <120> A siRNA targeting the HDAC3 gene of cotton bollworm and its application in pest control <160> 14 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <223> Primer HDAC3-F <400> 1 ccgttgaaag aagggattga 20 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <223> Primer HDAC3-R <400> 2 gcgttgatag cgagaagca 19 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <223> Primer RPS15-F <400> 3 ctgaggtcga tgaaactctc 20 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <223> Primer RPS15-R <400> 4 ctccatgagt tgctcattg 19 <210> 5 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-1 positive strand <400> 5 aaggaaccac caguugagaa c 21 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-1 antisense strand <400> 6 guucucaacu ggugguuccu u 21 <210> 7 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-2 positive strand <400> 7 aagaacgaau ucuaugaaga c 21 <210> 8 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-2 antisense strand <400> 8 gucuucauag aauucguucu u 21 <210> 9 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-3 positive strand <400> 9 aacugguccg gaggucuaca u 21 <210> 10 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-3 antisense strand <400> 10 auguagaccu ccggaccagu u 21 <210> 11 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-4 positive strand <400> 11 aacaugugua cgacaaccuc a 21 <210> 12 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-4 antisense strand <400> 12 ugagguuguc guacacaugu u 21 <210> 13 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-5 positive strand <400> 13 aaaccauaua gagccagugc u 21 <210> 14 <211> twenty one <212> RNA <213> Artificial sequence <223> siRNA-5 antisense strand <400> 14 agcacuggcu cuauaugguu u 21

Claims

1. A siRNA targeting the HDAC3 gene of cotton bollworm, characterized in that: The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

14.

2. Use of the siRNA according to claim 1 in regulating the growth, development or metamorphosis of cotton bollworm.

3. The use according to claim 2, characterized in that: The siRNA inhibits normal pupation or emergence of cotton bollworm, reduces the normal pupation rate or emergence rate, causes the cotton bollworm to be unable to pupate normally or delays the pupation time, or causes the cotton bollworm to have abnormal wings and be unable to fly normally after developing into an adult.

4. The use according to claim 2, characterized in that: The siRNA inhibits the egg laying or hatching of cotton bollworm, resulting in a significant reduction in the egg laying rate or egg hatching rate of cotton bollworm.

5. Use of the siRNA according to claim 1 in inhibiting the expression of HDAC3 gene of cotton bollworm, or in preventing and controlling cotton bollworm, or in preparing an insecticide for cotton bollworm.

6. The use according to claim 2 or 5, characterized in that: The application is to introduce the siRNA described in claim 1 into cotton bollworm, thereby inhibiting the normal pupation, eclosion, egg laying or hatching of cotton bollworm, thereby achieving the prevention and control of cotton bollworm.

7. The use according to claim 6, characterized in that: The introduction is to prepare the siRNA into a solution and then introduce it into the intestine of the cotton bollworm larvae.

8. The use according to claim 7, characterized in that: The siRNA solution was injected into the abdominal intersegmental membrane between the 6th and 7th segments of the cotton bollworm larvae.

9. The use according to claim 7 or 8, characterized in that: The cotton bollworm larvae are 6th instar larvae, the injection concentration of the siRNA solution is 2-4 ug / uL, and the injection volume is 2-4 uL.

10. The use according to claim 9, characterized in that: The injection concentration of siRNA solution was 3ug / uL, and the injection volume was 3uL.

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