CN115074362A - SiRNA targeting Helicoverpa armigera HDAC3 gene and application thereof in pest control - Google Patents
SiRNA targeting Helicoverpa armigera HDAC3 gene and application thereof in pest control Download PDFInfo
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Abstract
The invention discloses siRNA of a targeted cotton bollworm HDAC3 gene, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.13, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 14. Also discloses the application of the siRNA in the aspect of regulating the growth, development or metamorphosis of the cotton bollworm, the application in inhibiting the expression of the HDAC3 gene of the cotton bollworm, or the application in preventing and treating the cotton bollworm, or the application in the aspect of preparing a cotton bollworm insecticide. Selection of sirnas to silence cotton bollworm target genes is a more accurate and efficient approach than dsRNAs. One of the five siRNAs has outstanding silencing efficiency on HDAC3 gene, obvious inhibition effect on pupation, eclosion, oviposition and hatching of cotton bollworms and outstanding interference effect, is expected to be applied to field cotton bollworm control, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to siRNA targeting cotton bollworm HDAC3 gene and application thereof in pest control.
Background
Helicoverpa armigera (Hubner), Lepidoptera and Spodoptera are widely distributed in China and all over the world, are omnivorous agricultural pests, host over 300 species mainly damage crops such as cotton, corn, wheat, barley, tomatoes, green peppers and melons, and cause great loss of agricultural economy. Chemical insecticides and Bt insect-resistant cotton which are already available in the market at present can easily cause the problems of pesticide pollution, cotton bollworm resistance and the like, so that the development of a novel pest control method is particularly important.
Histone Deacetylases (HDACs) are a class of proteases and play an important role in the structural modification of chromosomes and the regulation of gene expression. HDACs tightly bind to negatively charged DNA, causing chromatin to compact and curl, thereby inhibiting transcription of genes. In the nucleus, histone acetylation and histone deacetylation processes are in dynamic equilibrium and are regulated by histone acetyltransferase and histone deacetylase together. HDAC3 belongs to class i HDACs, is ubiquitous and located in the nucleus. HDAC3 affects the expression of genes encoding proteins involved in the regulation of insect growth, development and metamorphosis by affecting the level of acetylation of histones. RNAi-mediated silencing of HDAC3 in drosophila S2 cells can lead to cell growth inhibition and disruption of sox14, and ecdysone genes eip74ef, nvy, etc.; larvae of tribolium castaneum, RNAi-mediated knock-out of HDAC3 gene affected their development, resulting in abnormal folding of pupae and adult wings.
siRNA (Small interfering RNA) is a double-stranded RNA with the length of 20 to 25 nucleotides, and is a gene silencing phenomenon that the restriction selection of a target point is realized through base complementary pairing and a specific gene is directly targeted, so that the efficient and specific degradation of mRNA is caused. siRNA is formed by cutting dsRNA by RNaseIII (such as Dicer) in cells, wherein the dsRNA can be exogenous, such as virus RNA replication intermediate or artificially introduced dsRNA; or endogenous, such as dsRNA formed by single-stranded RNA in a cell under the action of an RNA-dependent RNA polymerase. In plants, a natural RNA interference (RNAi) phenomenon exists, and Fire and the like firstly discover the effective and specific genetic interference of exogenous double-stranded RNA (dsRNA) on caenorhabditis elegans. The method is mastered by insect researchers, and the functions of various insect genes are gradually determined by introducing dsRNA to inhibit the expression of endogenous genes in insects so as to research the functions of the genes according to the phenotype of gene deletion. Injection of dsRNA into the body of insects is one of the more widely used pest control methods. However, FU J (Resistance to RNA interference by Plant-derived double-stranded RNAs) but Plant Cell and Environment, 2022, 45(6):1930-1941. DOI:10.1111/pce.14314.) and the like found that dsRNase activity in cotton bollworm intestinal fluid was high, and that the ingested dsRNA was rapidly degraded in intestinal fluid, on the contrary, siRNA was relatively stable in the digestive system, which is a major cause of the inefficiency of cotton bollworm dsRNA target genes, and these results indicated that Plant-derived siRNAs might trigger RNAi pathways of lepidoptera insects more effectively than dsRNAs, which would help to optimize the strategy of Plant-mediated RNAi pest control.
Disclosure of Invention
The invention aims to solve the problems and provides an siRNA targeting Helicoverpa armigera HDAC3 gene, wherein the nucleotide sequence of a sense strand of the siRNA is shown as SEQ ID NO.13, and the nucleotide sequence of an antisense strand of the siRNA is shown as SEQ ID NO. 14.
Another object of the present invention is to provide the use of the above siRNA for the regulation of the growth, development or metamorphosis of Helicoverpa armigera.
In the above application technical scheme, the siRNA inhibits normal pupation or eclosion of the cotton bollworm, reduces the normal pupation rate or eclosion rate, causes the cotton bollworm to fail to pupate normally or the pupation time is delayed, or causes the cotton bollworm to develop into an adult and then the wings to fail to fly normally.
In the application technical scheme, the siRNA inhibits the cotton bollworm from laying eggs or hatching, so that the egg laying rate or egg hatching rate of the cotton bollworm is obviously reduced.
The invention also aims to provide the application of the siRNA to inhibiting the expression of the cotton bollworm HDAC3 gene, or to control the cotton bollworm, or to prepare the cotton bollworm insecticide.
In the application technical scheme, the siRNA is introduced into the cotton bollworm, so that normal pupation, eclosion, oviposition or hatching of the cotton bollworm is inhibited, and further the prevention and control of the cotton bollworm are realized.
In the above application technical scheme, the introducing is to introduce the siRNA into the body of the cotton bollworm after preparing the siRNA into a solution, preferably into the intestinal tract of the cotton bollworm in an injection mode, and preferably, the cotton bollworm is a larva.
Preferably, the siRNA solution is injected into the interspinous membrane of sections 6 and 7 of Helicoverpa armigera.
Preferably, the cotton bollworm is 6-instar larva, the injection concentration of the siRNA solution is 2-4ug/uL, and the injection volume is 2-4 uL.
In the above application technical scheme, the injection concentration of the siRNA solution is 3ug/uL, and the injection volume is 3 uL.
The invention has the beneficial effects that: compared with dsRNAs, the method for silencing the cotton bollworm target gene by selecting the siRNA is a more accurate and effective method, and the siRNA has the advantages of specificity, high efficiency, high stability and the like. One of the five siRNAs has outstanding silencing efficiency on HDAC3 gene, obvious inhibition effect on pupation, eclosion, oviposition and hatching of cotton bollworms and outstanding interference effect, is expected to be applied to field cotton bollworm control, and has good application prospect.
Drawings
FIG. 1 is an analysis diagram of the expression pattern of HDAC3 gene at each spatiotemporal site of Helicoverpa armigera.
FIG. 2 shows the results of the silencing efficiency test of five siRNA injections at 12 h.
FIG. 3 is a graph of the analysis of silencing efficiency at various times after siHaHDAC3 injection.
FIG. 4 is a graph comparing pupation of Helicoverpa armigera after siHaHDAC3 injection.
FIG. 5 is a graph comparing the eclosion of Helicoverpa after siHaHDAC3 injection.
FIG. 6 is a graph comparing the egg production of Helicoverpa armigera after siHaHDAC3 injection.
FIG. 7 is a graph comparing the hatching of Heliothis armigera after siHaHDAC3 injection.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological reagents used are conventional reagents in the technical field unless otherwise specified.
Example 1
1. Amplification primer design
A cDNA sequence of the cotton bollworm HDAC3 gene (HaHDAC3) is obtained by using an online program such as NCBI Blast and the like, and the NCBI accession number is KM 983338.1.
Based on the found sequences, primers for amplification of the Helicoverpa armigera HDAC3 gene were designed using primer design software, and for amplification of the reference gene (RPS15, GenBank: XM-021326200.1), the primer sequences are shown in Table 1.
TABLE 1
2. Test insects
The cotton bollworm is bred by artificial feed indoors in Zhengzhou university laboratories, the breeding temperature is 26 +/-1 ℃, the relative humidity is 60 +/-10%, and the illumination period is as follows: 16h light, 8h dark.
3. Synthesis of first Strand cDNA
Extracting total RNA of cotton bollworm and using reagent kit
Cell/Tissue Total RNA Isolation Kit (Vazyme Nanjing China) extracts Total RNA from cotton bollworm Eggs (EG), 1-6-year-old (L1-L6) larvae, prepupa (PP), pupae (P), Female Adults (FA), Male Adults (MA), and 3-6-year-old cotton bollworm heads (H), integument (I), intestinal tract (H), and Mariotte (T). Use of
III 1st Strand cDNA Synthesis Kit (+ Gdnawind) (Vazyme Nanjing, China), first Strand cDNA was synthesized using the extracted total RNA.
4. Amplification of the bollworm HDAC3 Gene
The RT-qPCR reaction was performed using the previously obtained cDNA as a template, and ChamQ Universal SYBR qPCR Master Mix (Vazyme Nanjing China) and gene-specific primers (Table 1). RPS15 was used as a reference gene to correct for differences in sample cDNA. The total PCR reaction system is 20 ul: upstream and downstream primers at a concentration of 10pmol/ul each 0.4ul, cDNA template 2ul, 2 XChamQ Universal SYBR qPCR Master Mix 10ul, and ddH in balance 2 And O. The qPCR reaction procedure was as follows:
the results are shown in FIG. 1, including L1, L2, L3, L4, L5, L6, Egg (EG), prepupa (PP), pupa (P), Female Adult (FA), Male Adult (MA), head (3H), cuticle (3I), midgut (3H), Marsdenia tube (3T), head (4H), cuticle (4I), midgut (4H), Marsdenia tube (4T) of L4, head (5H), cuticle (5I), midgut (5H), Marsdenia tube (5T), head (6H), cuticle (6I), midgut (6H), and Marsdenia tube (6T) of L6. The expression level of HDAC3 gene in the above tissues is shown in FIG. 1, and the results show that HDAC3 gene is expressed in all age stages and tissues of Helicoverpa armigera, wherein the expression level of HDAC3 gene in 6 th larva and tissues is relatively highest, so 6 th Helicoverpa armigera is selected as the age stage of injection of siHaHDAC 3.
5. Synthesis of siRNA
Other sequences of the helicoverpa armigera HDAC family, including HDAC6, HDAC7, HDAC8, HDAC11, were searched for by nucleotide sequence BLAST alignment at the NCBI and ensembl isogenic databases, and these sequences were aligned to HDAC3, and the nucleotide sequence of the domain specific to HDAC3 gene was selected, five siRNA sequences were designed, and the synthesis was entrusted to shanghai bio-company. Five siRNA sequences were named: siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-5.
The sequences of the siRNA are respectively as follows:
siRNA-1: sense strand: 5'-AAGGAACCACCAGUUGAGAAC-3' (SEQ ID NO.5),
antisense strand: 5'-GUUCUCAACUGGUGGUUCCUU-3' (SEQ ID NO. 6).
siRNA-2: a sense strand: 5'-AAGAACGAAUUCUAUGAAGAC-3' (SEQ ID NO.7),
antisense strand: 5'-GUCUUCAUAGAAUUCGUUCUU-3' (SEQ ID NO. 8).
siRNA-3: sense strand: 5'-AACUGGUCCGGAGGUCUACAU-3' (SEQ ID NO.9),
antisense strand: 5'-AUGUAGACCUCCGGACCAGUU-3' (SEQ ID NO. 10).
siRNA-4: sense strand: 5'-AACAUGUGUACGACAACCUCA-3' (SEQ ID NO.11),
antisense strand: 5'-UGAGGUUGUCGUACACAUGUU-3' (SEQ ID NO. 12).
siRNA-5: a sense strand: 5'-AAACCAUAUAGAGCCAGUGCU-3' (SEQ ID NO.13), antisense strand: 5'-AGCACUGGCUCUAUAUGGUUU-3' (SEQ ID NO. 14).
6. RNA interference
The five synthesized siRNAs were diluted with DEPC water to a final concentration of 3ug/ul, respectively, as a treatment group, and a control group was injected with DEPC water.
The 6 th-age cotton bollworms with uniform size and consistent health status are selected and subjected to micro-injection on the ventral internode membranes of the 6 th section and the 7 th section of the cotton bollworms, the injection volume of each cotton bollworm is 3ul, the injection concentration is 3ug/ul, and 100 cotton bollworms are respectively injected into the treatment group and the control group. After the injection is finished, the cotton bollworm larvae are placed in an artificial feed box for culturing, the feeding temperature is 26 +/-1 ℃, the relative humidity is 60 +/-10%, and the illumination period is as follows: 16h light, 8h dark.
Samples of the treatment group and the control group are taken 12h, 24h, 36h and 48h after the injection is finished, the silencing efficiency of the HDAC3 gene is detected, and the RPS15 is used as a difference result of the internal reference gene correction sample cDNA. The detection method is RT-qPCR, and the primers, the PCR reaction system and the amplification program are the same as those in the previous RT-qPCR method for amplifying the cotton bollworm HDAC3 gene.
The results of the silencing efficiency detection 12h after five siRNA injections are shown in FIG. 2, the silencing efficiency of siRNA-4 and siRNA-5 is good, especially the silencing efficiency of siRNA-5 is outstanding, and siRNA-5 is selected for injection experiments subsequently.
The silencing efficiency after 12h after siRNA-5 injection reaches more than 90%, and the silencing efficiency can be maintained within 48h, and the results are shown in FIG. 3, and compared with the control group, the silencing efficiencies of 12h, 24h, 36h and 48h are respectively 99.6%, 99.1%, 99.4% and 94.4%.
After siRNA-5 injection, the growth, pupation, eclosion, oviposition and hatching conditions of the cotton bollworms are observed: the cotton bollworm in the siRNA-5 treated group failed to pupate normally or the pupation time was delayed, and the CK group could pupate normally, as shown in FIG. 4, the abnormal pupation rate reached 21%. After eclosion, the cotton bollworm in the treatment group developed into adult with abnormal rear wing, which failed to fly normally, and the abnormal wing rate reached 28%, while the cotton bollworm in the control group developed into adult with normal wing, as shown in fig. 5. After spawning, counting the number of spawned eggs, wherein the number of the spawned eggs of the cotton bollworms in the treated group is 39% of that in the control group, and the spawning condition is shown in figure 6; hatching rates of eggs, 50 eggs which are just hatched in the treatment group and the control group are respectively selected and placed in a 96-well plate, and after 96 hours, the hatching rates are counted, wherein the hatching rate of the treatment group is 6 percent, the hatching rate of the control group is 82 percent, and the hatching condition is shown in fig. 7. Therefore, HDAC3 gene plays an important role in pupation, eclosion, oviposition and hatching of cotton bollworm.
By combining the experimental results, the siRNA for inhibiting the cotton bollworm growth and development related genes is injected into the intestinal tract of the bollworm, so that the expression of the target genes can be inhibited, and the growth and development of pests are influenced, thereby achieving the purpose of pest control.
Sequence listing
<110> Zhengzhou university
<120> siRNA targeting Helicoverpa armigera HDAC3 gene and application thereof 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> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-1 sense strand
<400> 5
aaggaaccac caguugagaa c 21
<210> 6
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-1 antisense strand
<400> 6
guucucaacu ggugguuccu u 21
<210> 7
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-2 sense strand
<400> 7
aagaacgaau ucuaugaaga c 21
<210> 8
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-2 antisense strand
<400> 8
gucuucauag aauucguucu u 21
<210> 9
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-3 sense strand
<400> 9
aacugguccg gaggucuaca u 21
<210> 10
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-3 antisense strand
<400> 10
auguagaccu ccggaccagu u 21
<210> 11
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-4 sense strand
<400> 11
aacaugugua cgacaaccuc a 21
<210> 12
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-4 antisense strand
<400> 12
ugagguuguc guacacaugu u 21
<210> 13
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-5 sense strand
<400> 13
aaaccauaua gagccagugc u 21
<210> 14
<211> 21
<212> RNA
<213> Artificial sequence
<223> siRNA-5 antisense strand
<400> 14
agcacuggcu cuauaugguu u 21
Claims (10)
1. An siRNA targeting a cotton bollworm HDAC3 gene, characterized in that: the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 14.
2. Use of the siRNA of claim 1 for the modulation of the growth, development or metamorphosis of Heliothis armigera.
3. Use according to claim 2, characterized in that: the siRNA inhibits normal pupation or eclosion of the cotton bollworm, reduces the normal pupation rate or eclosion rate, causes the cotton bollworm to be incapable of pupating normally or the pupation time is delayed, or causes the cotton bollworm to develop into an adult and then wings to be abnormal and incapable of flying normally.
4. Use according to claim 2, characterized in that: the siRNA inhibits the cotton bollworm from laying eggs or hatching, so that the egg laying rate or egg hatching rate of the cotton bollworm is obviously reduced.
5. The use of the siRNA of claim 1 for inhibiting the expression of Helicoverpa armigera HDAC3 gene, or for controlling Helicoverpa armigera, or for preparing Helicoverpa armigera insecticide.
6. Use according to claim 2 or 5, characterized in that: the use is to introduce the siRNA described in claim 1 into cotton bollworm, thereby achieving inhibition of normal pupation, eclosion, oviposition or hatching of cotton bollworm, and further achieving control of cotton bollworm.
7. Use according to claim 6, characterized in that: the introduction is to prepare siRNA into a solution and then introduce the siRNA into the body of the cotton bollworm, preferably to introduce the siRNA into intestinal tracts of the cotton bollworm in an injection mode, and preferably to select the cotton bollworm as a larva.
8. Use according to claim 7, characterized in that: the siRNA solutions were injected into the interspinous membrane of sections 6 and 7 of Helicoverpa armigera.
9. Use according to claim 7 or 8, characterized in that: the cotton bollworm is 6-instar larva, the injection concentration of the siRNA solution is 2-4ug/uL, and the injection volume is 2-4 uL.
10. Use according to claim 9, characterized in that: the injection concentration of the siRNA solution was 3ug/uL, and the injection volume was 3 uL.
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|---|---|---|---|---|
| US20060272049A1 (en) * | 2003-11-17 | 2006-11-30 | Waterhouse Peter M | Insect resistance using inhibition of gene expression |
| CN108949769A (en) * | 2018-07-24 | 2018-12-07 | 江西农业大学 | A kind of cotton bollworm molt hormone regulating and controlling factor E78-C gene cDNA and its application |
| CN108949770A (en) * | 2018-07-24 | 2018-12-07 | 江西农业大学 | Bollworm E75 gene and its control of insect application mediated in RNA |
| AU2020102815A4 (en) * | 2020-10-16 | 2020-12-10 | Shihezi University | RNAi Technology-Based Helicoverpa Amigera Lethal Gene and Bioinsecticidal Formulation Thereof |
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2022
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| US20060272049A1 (en) * | 2003-11-17 | 2006-11-30 | Waterhouse Peter M | Insect resistance using inhibition of gene expression |
| CN108949769A (en) * | 2018-07-24 | 2018-12-07 | 江西农业大学 | A kind of cotton bollworm molt hormone regulating and controlling factor E78-C gene cDNA and its application |
| CN108949770A (en) * | 2018-07-24 | 2018-12-07 | 江西农业大学 | Bollworm E75 gene and its control of insect application mediated in RNA |
| AU2020102815A4 (en) * | 2020-10-16 | 2020-12-10 | Shihezi University | RNAi Technology-Based Helicoverpa Amigera Lethal Gene and Bioinsecticidal Formulation Thereof |
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| Title |
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