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CN116875612A - Apolygus lucorum CtsL gene, and corresponding dsRNA sequence and application thereof - Google Patents

Apolygus lucorum CtsL gene, and corresponding dsRNA sequence and application thereof Download PDF

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CN116875612A
CN116875612A CN202311087404.4A CN202311087404A CN116875612A CN 116875612 A CN116875612 A CN 116875612A CN 202311087404 A CN202311087404 A CN 202311087404A CN 116875612 A CN116875612 A CN 116875612A
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ctsl
gene
lygus lucorum
dsrna
sequence
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梁思佳
金双侠
李婉宁
刘军和
王福秋
李波
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Huazhong Agricultural University
Huanghuai University
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Huanghuai University
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Abstract

The invention relates to the technical field of plant genetic engineering. The invention provides a lygus lucorum CtsL gene, a corresponding dsRNA sequence and application thereof, wherein the nucleotide sequence of the lygus lucorum CtsL gene is shown in SEQ ID NO: 1. The invention also comprises a dsRNA sequence of the targeting lygus lucorum CtsL gene, and the sequence has good silencing effect on the lygus lucorum CtsL gene. Injection of the dsRNA sequences can significantly reduce survival rate of lygus lucorum. Therefore, the CtsL gene and the lethal dsRNA sequence thereof provided by the invention can be used for the development of transgenic insect-resistant plants or novel pesticides.

Description

Apolygus lucorum CtsL gene, and corresponding dsRNA sequence and application thereof
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a lygus lucorum CtsL gene, a corresponding dsRNA sequence and application thereof.
Background
Lygus lucorum Adelphocoris suturalis (taxonomically Hemiptera: apolygus lucorum) is an important multi-feeding agricultural pest, mainly damaging cotton, soybean, alfalfa, grape, date, etc. In recent years, with the popularization of transgenic Bt cotton, the application times and the application amount of chemical pesticides in cotton fields are reduced, so that the population quantity of field plant bugs is greatly increased, the hazard is aggravated, and the method has become a great problem for damaging the production of various crops. The control of lygus lucorum is currently based on the application of chemical pesticides. Because of the environmental pressure and drug resistance caused by the long-term use of chemical pesticides in large quantity, the development and utilization of pollution-free control measures conforming to the concepts of environmental protection, health and sustainable development become the current control hot spot.
RNA interference (RNAinterference, RNAi) is an important gene silencing technology discovered in recent years, and is a phenomenon that homologous RNA (double-stranded ribonucleic acid, dsRNA) is efficiently and specifically degraded by double-stranded RNA. Since the RNAi technology can specifically inhibit the expression of specific genes, the technology has been widely used for targeting the expression of genes essential to interfering pests, so that the adaptability of the pests is reduced, even the pests die, and finally the purpose of pest control is achieved. The method has the advantages of no need of adding chemical pesticides, no need of synthesizing proteins, multiple target gene selectivities (any essential genes), capability of superposing multiple target genes (preventing target pests from generating resistance to a single gene), and the like. However, the first step in insect resistance using RNAi technology is to screen for potent lethal genes.
Disclosure of Invention
The invention aims to provide a lygus lucorum CtsL gene, a corresponding dsRNA sequence and application thereof, wherein an essential gene-CtsL gene for the lygus lucorum is obtained through transcriptome data screening, a dsRNA sequence targeting the gene is synthesized in vitro, and the dsRNA sequence is proved to be capable of silencing the lygus lucorum CtsL gene with high efficiency through in vitro injection experiments, so that the survival rate of nymphs is remarkably reduced. Solves the problem that no effective insecticidal dsRNA exists for the lygus lucorum at home and abroad at present, and has good application prospect in the aspects of developing novel pesticides and insect-resistant plants.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a lygus lucorum CtsL gene, the nucleotide sequence of which is shown in SEQ ID NO:1 is shown as follows:
ATGAAGACTGTTCTCGCTTTCGCGTGTCTGGTGGCCCTCGGGCTCGCCATGCCGATGTCCGACGACGCCGAGTGGGAAAGCTACAAGGCCAAGTACGGAAAGAACTACGAGTCCAACGAACATGAATCTCTCAGGAGAACCATCTACTTCACCGCCAAGGAAAAAGTCCAGGAGCACAACGCCCGCTTCGAACAGGGTCTGGTCTCCTACAAACTGGGATTGAACCGATTCGCTGACATGCTCAACGGTGAATTCCGTAAGATGATGAACGGCTACAGGAGAGGCACCCCCAGGAACTCTTTCTCCTTCCACGTTGAATCCAACGTAACCCTCCCAGCCACGGTTGACTGGAGAACGAAAGGAGCCGTCACCCCCATCAAGAACCAGGGACAGTGCGGATCTTGCTGGGCTTTCAGTACCACCGGATCTCTTGAGGGACAACACGCTCTGAAGAAGGGAAAACTCGTATCTCTCAGTGAACAGCAACTCGTCGACTGCTCAAGCGCCGAAGGAAACGATGGATGCGACGGTGGTCTTATGGACAACGGTTTCGCTTACATCAAGGCCAACAACGGAATCGACACTGAAGCTTCCTACCCTTACACCGGTGAAGACGGTACCTGCGCTGCCAAGAAGAGTGACATTGCTGCCACCGTCACTGGACACGTAGATATCAAGTCTGGTAGCGAATCTAATCTTCAAGACGCTTCTGCCACTGTCGGACCAATCTCTGTAGCTATTGATGCCAGCAGCTATGACTTCCAACTCTACGAAAGCGGAGTTTACGACGAGAGCGACTGCAGCACCACCGAGCTGGATCACGGTGTCTTGGTTGTTGGATACGGAACCGACTCCGGATCCGCCTACTGGCTCGTCAAGAACTCTTGGGGTACCGACTGGGGTATTAATGGATACATCCAGATGAGCAGAAACAAGGACAACCAGTGCGGTATCGCCACTGAGGCCAGCTACCCACTCGTCTAA。
preferably, the amino acid sequence of the lygus lucorum CtsL gene is shown in SEQ ID NO:2 is shown as follows:
MKTVLAFACLVALGLAMPMSDDAEWESYKAKYGKNYESNEHESLRRTIYFTAKEKVQEHNARFEQGLVSYKLGLNRFADMLNGEFRKMMNGYRRGTPRNSFSFHVESNVTLPATVDWRTKGAVTPIKNQGQCGSCWAFSTTGSLEGQHALKKGKLVSLSEQQLVDCSSAEGNDGCDGGLMDNGFAYIKANNGIDTEASYPYTGEDGTCAAKKSDIAATVTGHVDIKSGSESNLQDASATVG PISVAIDASSYDFQLYESGVYDESDCSTTELDHGVLVVGYGTDSGSAYWLVKNSWGTDWGINGYIQMSRNKDNQCGIATEASYPLV。
the invention also provides application of the lygus lucorum CtsL gene as a lygus lucorum control target.
The invention also provides application of the lygus lucorum CtsL gene in cultivation of lygus lucorum resistant plants.
The invention also provides a primer pair for preparing dsRNA targeting the lygus lucorum CtsL gene, wherein the primer pair comprises an upstream primer dsCtsL-F and a downstream primer dsCtsL-R;
the nucleotide sequence of the upstream primer dsCtsL-F is shown in SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the downstream primer dsCtsL-R is shown in SEQ ID NO: 4.
The invention also provides application of the primer pair in preparation of dsRNA sequence of targeting lygus lucorum CtsL gene.
The invention also provides a dsRNA sequence of the targeting lygus lucorum CtsL gene, wherein the dsRNA sequence is shown in SEQ ID NO:7, as follows:
CTTGAGGGACAACACGCTCTGAAGAAGGGAAAACTCGTATCTCTCAGTGAACAGCAACTCGTCGACTGCTCAAGCGCCGAAGGAAACGATGGATGCGACGGTGGTCTTATGGACAACGGTTTCGCTTACATCAAGGCCAACAACGGAATCGACACTGAAGCTTCCTACCCTTACACCGGTGAAGACGGTACCTGCGCTGCCAAGAAGAGTGACATTGCTGCCACC。
the invention also provides application of the dsRNA sequence of the targeting lygus lucorum CtsL gene in lygus lucorum control.
The invention also provides a primer pair for cloning the lygus lucorum CtsL gene, which comprises an upstream primer CtsL-F and a downstream primer CtsL-R;
the nucleotide sequence of the upstream primer CtsL-F is shown in SEQ ID NO:5 is shown in the figure;
the nucleotide sequence of the downstream primer CtsL-R is shown in SEQ ID NO: shown at 6.
The invention also provides application of the primer pair in cloning of the lygus lucorum CtsL gene.
The invention provides a lygus lucorum CtsL gene, a corresponding dsRNA sequence and application thereof, wherein the nucleotide sequence of the lygus lucorum CtsL gene is shown in SEQ ID NO: 1. The invention also comprises a dsRNA sequence of the targeting lygus lucorum CtsL gene, and the sequence has good silencing effect on the lygus lucorum CtsL gene. Injection of the dsRNA sequences can significantly reduce survival rate of lygus lucorum. Therefore, the CtsL gene and the lethal dsRNA sequence thereof provided by the invention can be used for the development of transgenic insect-resistant plants or novel pesticides.
Drawings
FIG. 1 is a survival curve of lygus lucorum after injection of dsRNA targeting the CtsL gene, where "dsCtsL" represents the treatment group injected with the CtsL gene dsRNA; "dsGFP" means control group injected with dsRNA of GFP gene, "x" means p <0.05; ", denotes p <0.001; "ns" means that p >0.05.
FIG. 2 is a different phenotype of lygus lucorum after injection of dsRNA targeting the CtsL gene, where "dsCtsL" represents the treated group injected with the CtsL gene dsRNA; "dsGFP" refers to the control group injected with GFP gene dsRNA.
Fig. 3 is the expression level of CtsL gene after injection of dsRNA targeting CtsL gene, wherein "×" indicates p <0.05; ", denotes p <0.001; "ns" means that p >0.05; "dsCtsL" means the treatment group injected with dsRNA of CtsL gene; "dsGFP" refers to the control group injected with GFP gene dsRNA.
Detailed Description
The invention provides a lygus lucorum CtsL gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.
In the invention, the amino acid sequence of the lygus lucorum CtsL gene is shown in SEQ ID NO: 2.
The invention also provides application of the lygus lucorum CtsL gene as a lygus lucorum control target.
The invention also provides application of the lygus lucorum CtsL gene in cultivation of lygus lucorum resistant plants.
The invention also provides a primer pair for preparing dsRNA targeting the lygus lucorum CtsL gene, wherein the primer pair comprises an upstream primer dsCtsL-F and a downstream primer dsCtsL-R;
the nucleotide sequence of the upstream primer dsCtsL-F is shown in SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the downstream primer dsCtsL-R is shown in SEQ ID NO: 4.
The invention also provides application of the primer pair in preparation of dsRNA sequence of targeting lygus lucorum CtsL gene.
The invention also provides a dsRNA sequence of the targeting lygus lucorum CtsL gene, wherein the dsRNA sequence is shown in SEQ ID NO: shown at 7.
The invention also provides application of the dsRNA sequence of the targeting lygus lucorum CtsL gene in lygus lucorum control.
The invention also provides a primer pair for cloning the lygus lucorum CtsL gene, which comprises an upstream primer CtsL-F and a downstream primer CtsL-R;
the nucleotide sequence of the upstream primer CtsL-F is shown in SEQ ID NO:5 is shown in the figure;
the nucleotide sequence of the downstream primer CtsL-R is shown in SEQ ID NO: shown at 6.
The invention also provides application of the primer pair in cloning of the lygus lucorum CtsL gene.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 cloning of the lygus lucorum CtsL Gene
(1) Extraction of lygus lucorum total RNA: 20mg of lygus lucorum sample was weighed and placed in a 1.5ml enzyme-free tube, and after sufficient grinding with a disposable enzyme-free grinding rod and liquid nitrogen, total RNA was extracted using the SV total RNA isolation system extraction kit from Promega corporation, the detailed procedure being referred to the kit instructions.
(2) Synthesis of cDNA: the total RNA extracted in the above step (1) was synthesized into a cDNA template using a PrimeScriptTM RT Master Mix reverse transcription kit (see the kit instructions for details).
(3) Primer design: the nucleic acid sequence of the CtsL gene (see SEQ ID NO: 1) is obtained by transcriptome sequencing, and the primers are designed to verify the predicted open reading frame. The primer sequences designed and synthesized were as follows:
the upstream primer CtsL-F:5'-CCATTAATTTCACCTCTTTGTGGCA-3' (SEQ ID NO: 5),
the downstream primer CtsL-R:5'-GTGCGTTTAGACGAGTGGGT-3' (SEQ ID NO: 6).
The above primers were synthesized by Shanghai Biotechnology services Co.
(4) And (3) PCR amplification: PCR amplification was performed using the above primers CtsL-F and CtsL-R, and the PCR system was referred to the Ex Taq enzyme instructions of Takara Shuzo Co., ltd. PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 2min,38 cycles; extending at 72℃for 10min. After amplification, the target fragment was identified by 1% agarose gel electrophoresis, excised and purified and recovered using the AxyGen DNA gel recovery kit.
(5) Cloning of PCR products: the PCR product was connected to pEASY-T1 vector (see kit description) according to the instruction using pEASY-T1 Simple Cloning Kit from TransGen, and the positive clones were screened into competent cells of E.coli DH 5. Alpha. And sent to Beijing qingke new industry Biotechnology Co., ltd for sequencing. Finally, the accuracy of the nucleotide sequence of the CtsL gene obtained by sequencing is verified by comparing the nucleotide sequence obtained by sequencing with the nucleotide sequence obtained by transcriptome sequencing by utilizing NCBI (https:// www.ncbi.nlm.nih.gov /), and the result shows that the sequencing result of the invention is consistent.
EXAMPLE 2dsRNA Synthesis
Preparation of dsrna templates:
(1) Based on the CtsL gene sequence obtained in example 1, dsRNA region was predicted by siDirect version 2.0 and specific amplification primers (5' -end plus T7promoter sequence gcgtaatacgactcactatagg) were designed for amplification of dsRNA fragment of CtsL gene using NCBIPrimer-BLAST (https:// www.ncbi.nlm.ni h. Gov/tools/primer-BLAST/index. Cgilink_loc=blasthome) as follows:
the upstream primer sequence dsCtsL-F: CTTGAGGGACAACACGCTCTG (SEQ ID NO: 3),
the downstream primer sequence dsCtsL-R: GGTGGCAGCAATGTCACTCTT (SEQ ID NO: 5).
PCR amplification was performed using the above primers dsCtsL-F and dsCtsL-R, and the PCR reaction system was referred to the Ex Taq enzyme Instructions of Takara Shuzo Co., ltd.
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, renaturation at 72 ℃ for 2min,38 cycles; extending at 72℃for 10min. After amplification, the target fragment was identified by 1% agarose gel electrophoresis, excised and purified and recovered using the AxyGen DNA gel recovery kit. Finally, the DNA was ligated to pEASY-T1 vector (see kit instructions) by TA cloning.
(2) The plasmid containing the target fragment was extracted using the AxyPrep Plasmid Miniprep kit kit of AxyGen, and the plasmid was used as a template, and the specific primers dsCtsL-F and dsCtsL-R were used for a second PCR amplification, and the PCR system and the reaction procedure were the same as those of (1) above.
Purification of dsRNA templates
The product of the second PCR was purified by phenol chloroform extraction, and the procedure was as follows:
(1) The material to be purified is treated with DEPC H 2 O constant volume to 300 μl (the system can be expanded in equal proportion according to actual needs).
(2) Equal volumes of phenol were added: chloroform: isoamyl alcohol (volume ratio of 25:24:1), after sufficient shaking, the mixture was centrifuged at 12000rpm at room temperature for 10min.
(3) The upper phase was taken at 250. Mu.l, placed in a fresh RNase-free 1.5ml centrifuge tube, 25. Mu.l of 3M sodium acetate (pH 5.2) and 550. Mu.l of absolute ethanol (-20 ℃ C. Pre-cooled) were added, gently mixed and then placed at-20 ℃ C. For precipitation for 3h.
(4) Centrifuge at 12000rpm for 15min at 4℃and discard supernatant.
(5) Adding 75% ethanol (with DEPC H) 2 O preparation, -20 ℃ pre-cooling) are reversed for several times, and the precipitate is washed.
(6) Centrifuge at 4℃at 7500rpm for 5min.
(7) The supernatant was discarded, the pellet was air-dried and 20. Mu.l DEPC H was added 2 O dissolves the precipitate.
(8) The recovered product was detected by 1% agarose gel electrophoresis and the product concentration and OD values were detected by NanoDrop 2000.
Dsrna synthesis:
(1) dsRNA synthesis reaction system
The synthesis reaction system of dsRNA is shown in Table 1.
TABLE 1dsRNA synthesis reaction System
Reagent(s) 50 mu L system
5×Transcription buffer 10μl
ATP，CTP，GTP，UTP(100mM) 2 μl each
RNase inhibitor (40U/. Mu.l) 1.5μl
Template 1μg
T7 RNA Polymerase(200U/μl) 0.5μl
DEPCH 2 O constant volume to 50μl
Flick and mix evenly, and centrifuge instantly. 37℃for 4 hours.
(2) DNAse I digests the DNA template and reagents as described in Table 2 are added in proportion.
TABLE 2DNAse I digestion reaction System
Mixing, instantaneous centrifuging at 37deg.C for 30min.
Dsrna purification:
(1) The material to be purified is treated with DEPC H 2 O constant volume to 300 μl (the system can be expanded in equal proportion according to actual needs).
(2) 150 μl of water-saturated phenol and 150 μl of chloroform were added, and after sufficient shaking, the mixture was centrifuged at 12000rpm at 4℃for 15min.
(3) The upper phase was taken at 250. Mu.l, placed in a fresh RNase-free 1.5ml centrifuge tube, 25. Mu.l of 3M sodium acetate (pH 5.2) and about 700. Mu.l of absolute ethanol (-20 ℃ C. Pre-cooled), gently mixed and allowed to stand at-20 ℃ C. Overnight.
(3) Centrifuge at 12000rpm for 30min at 4℃and discard supernatant.
(4) Adding 75% ethanol (DEPC H) 2 O preparation, -20 ℃ pre-cooling) are reversed for several times, and the precipitate is washed.
(5) Centrifuge at 4℃at 7500rpm for 5min.
(6) The supernatant was discarded, the pellet was air-dried and 20. Mu.l DEPC H was added 2 O dissolves the precipitate.
(7) The dsRNA mass was detected by 1% agarose gel electrophoresis and the product concentration and OD values were detected by NanoDrop 2000.
(8) The dsRNA was diluted to 10. Mu.g/. Mu.l for use.
EXAMPLE 3 Effect of dsRNA injected with CtsL Gene on expression level of lygus lucorum
The synthesized dsGFP is used as a control, and the synthesized dsRNA is injected into the body of the initial molt 3-year nymphs from the outermost sides of the posterior chest and the abdominal internode of lygus lucorum by using a microinjection instrument.
Survival of the surface insects was observed and recorded daily for 14 days, 80 insects were treated each, and repeated three times.
Collecting Apolygus lucorum at 2, 4, 6 and 8 days after injection, extracting total RNA, synthesizing cDNA, and using Takara Bio engineering Dain Limited companyPremix ExTaq TM II and Bio-Rad Detection iQ2 System. The expression level of the CtsL gene was detected.
Test results and analysis:
(1) Effect of dsRNA injected into CtsL Gene on lygus lucorum
Three biological replicates showed that the mortality of lygus lucorum injected with dsRNA of the CtsL gene was significantly higher than that of the control group (dsGFP) (FIG. 1, P < 0.05). In addition, we also observed that the lygus lucorum injected with the CtsL gene dsRNA had a death peak period of 8-11 d after injection, and this period was the eclosion period of the lygus lucorum, and the test insects all died due to failure to complete normal eclosion (see FIG. 2).
(2) Effect of dsRNA injected into CtsL Gene on expression of lygus lucorum CtsL Gene
The qRT-PCR detection result shows that: the expression level of CtsL in lygus lucorum was significantly lower in the control group (as in fig. 3, p < 0.05) at 2, 4, 6 and 8 days after injection of dsRNA of CtsL gene. Therefore, the interfering sequence of the lygus lucorum CtsL gene can obviously inhibit the expression of the CtsL gene.
The results show that injection of dsRNA targeting the CtsL gene can remarkably inhibit the expression of the CtsL gene and can effectively kill the lygus lucorum nymphs.
The dsRNA lethal sequence of the lygus lucorum provided by the invention can be expressed by bacteria or transgenic plants, is used for green prevention and control of the lygus lucorum, and has good application prospects in the aspect of developing novel pesticides.
From the above examples, the invention provides a lygus lucorum CtsL gene, a corresponding dsRNA sequence and application thereof, wherein the nucleotide sequence of the lygus lucorum CtsL gene is shown in SEQ ID NO: 1. The invention also comprises a dsRNA sequence of the targeting lygus lucorum CtsL gene, and the sequence has good silencing effect on the lygus lucorum CtsL gene. Injection of the dsRNA sequences can significantly reduce survival rate of lygus lucorum. Therefore, the CtsL gene and the lethal dsRNA sequence thereof provided by the invention can be used for the development of transgenic insect-resistant plants or novel pesticides.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The adelphocoris lucorum CtsL gene is characterized in that the nucleotide sequence of the adelphocoris lucorum CtsL gene is shown in SEQ ID NO: 1.
2. The lygus lucorum CtsL gene according to claim 1, wherein the amino acid sequence of the lygus lucorum CtsL gene is as set forth in SEQ ID NO: 2.
3. Use of the lygus lucorum CtsL gene according to claim 1 or 2 as a lygus lucorum control target.
4. Use of the lygus lucorum CtsL gene according to claim 1 or 2 for breeding a plant resistant to lygus lucorum.
5. A primer pair for preparing a dsRNA targeting the lygus lucorum CtsL gene of claim 1, characterized in that the primer pair comprises an upstream primer dsCtsL-F and a downstream primer dsCtsL-R;
the nucleotide sequence of the upstream primer dsCtsL-F is shown in SEQ ID NO:3 is shown in the figure;
the nucleotide sequence of the downstream primer dsCtsL-R is shown in SEQ ID NO: 4.
6. The use of the primer pair according to claim 5 for preparing dsRNA sequence targeting lygus lucorum CtsL gene.
7. A dsRNA sequence targeting the lygus lucorum CtsL gene, characterized in that the dsRNA sequence is as set forth in SEQ ID NO: shown at 7.
8. Use of the dsRNA sequence of targeting lygus lucorum CtsL gene according to claim 7 for the control of lygus lucorum.
9. A primer pair for cloning a lygus lucorum CtsL gene, which is characterized by comprising an upstream primer CtsL-F and a downstream primer CtsL-R;
the nucleotide sequence of the upstream primer CtsL-F is shown in SEQ ID NO:5 is shown in the figure;
the nucleotide sequence of the downstream primer CtsL-R is shown in SEQ ID NO: shown at 6.
10. Use of the primer pair of claim 9 in cloning of the lygus lucorum CtsL gene.
CN202311087404.4A 2023-08-28 2023-08-28 Apolygus lucorum CtsL gene, and corresponding dsRNA sequence and application thereof Pending CN116875612A (en)

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