CN113789329A - RNA preparation for inhibiting boring behavior of chilo suppressalis - Google Patents
RNA preparation for inhibiting boring behavior of chilo suppressalis Download PDFInfo
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Abstract
The invention provides an RNA preparation for inhibiting chilo suppressalis borer boring behavior, firstly, chilo suppressalis borer epidermal protein gene LCP16/17 is cloned from chilo suppressalis borer, primers are designed for chilo suppressalis epidermal protein gene LCP16/17, dsRNA for interfering chilo suppressalis epidermal protein gene LCP16/17 is synthesized, a system for delivering dsRNA by using a nano carrier is established, and the dsRNA is delivered into a worm body through dropping body surface to interfere chilo suppressalis epidermal protein gene LCP 16/17. The results show that: after the dsRNA is injected into the chilo suppressalis, the change of the boring behavior of the chilo suppressalis is found, the occurrence of the boring behavior is obviously reduced, and the dsRNA has an important effect on the prevention and the treatment of the chilo suppressalis. The invention provides a new method for molecular regulation of pests and also provides a theoretical basis for deeply understanding the growth and development mechanism of insects and creating a new biological pesticide preparation.
Description
Technical Field
The invention provides an RNA preparation for inhibiting chilo suppressalis borer boring behavior, particularly relates to synthesis and application of chilo suppressalis borer epidermal protein LCP16/17 gene and targeting dsRNA, and belongs to the technical field of agricultural biology.
Technical Field
Chilo suppressalisChilo suppressalisBelongs to lepidoptera, borer moth and eating rice stalks, is one of the most serious common pests on rice in China, and is mainly distributed in Yangtze river basin and rice areas in the south of the Yangtze river basin in China. Chemical control has been the main principle for a long time, but the use of a large amount of pesticides results in drug resistance and pesticide residuesThe problems of retention and the like are increasingly serious. Therefore, development of a novel and highly effective control method is urgently required.
The insect cuticle is the first barrier to prevent foreign body from losing water and protecting it from external pathogens and harmful substances. The chemical components of the epidermis are mainly lipid, protein and chitin, the epidermal protein exists in the upper epidermal layer and the primary epidermal layer, the upper epidermal layer is formed by crosslinking the lipid and the protein and does not contain the chitin, and the chitin exists only in the primary epidermal layer and is crosslinked with the epidermal protein to form the primary epidermal layer. Thus, epidermal proteins are a potential target site.
The RNA interference technology is a relatively common experimental technology, and is a hot direction for disease treatment and agricultural pest control by delivering dsRNA and specifically silencing a target gene to perform functional research on the gene. The nano material can carry dsRNA to break through the body wall, the gene interference efficiency is high, and the nano carrier is safe to non-target organisms. Therefore, the RNAi technology is adopted, the chilo suppressalis epidermal protein LCP16/17 is specifically silenced through dripping the body surface, the change of the chilo suppressalis boring behavior is found, the occurrence of the boring behavior is remarkably reduced, and the method plays an important role in preventing and treating the chilo suppressalis.
Disclosure of Invention
The invention aims to overcome the defects and provide an RNA preparation for inhibiting chilo suppressalis borer boring behavior, wherein the RNA preparation is prepared by firstly providing chilo suppressalis LCP16/17 gene, and then designing and synthesizing corresponding targeting dsRNA according to chilo suppressalis LCP16/17 gene. The dsRNA is mixed with a nano-carrier, larvae with consistent growth state are selected, and the dsRNA is delivered into the body by a dropping method. The result shows that the expression of the chilo suppressalis epidermal protein gene LCP16/17 at the mRNA level is obviously reduced, the boring behavior of larvae is obviously reduced, and the result provides a new method and a new idea for green prevention and control of chilo suppressalis.
The invention is realized by the following steps: an RNA preparation for inhibiting the boring behavior of chilo suppressalis, which is characterized by comprising the following steps:
searching in a chilo suppressalis transcriptome database by using a bioinformatics analysis method according to a known insect CPR family protein sequence conserved region (motif) to obtain an LCP16/17 gene fragment;
step (2), designing an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4 according to the known LCP16/17 gene fragment by using primer premier 5.0 software;
SEQ ID NO.3:ATGAAATCGATCGTATTAAT；
SEQ ID NO.4:TTATTGGACCTTTGGGATGG；
extracting total RNA of chilo suppressalis third-instar larvae and performing reverse transcription to obtain a cDNA template;
step (4), obtaining a Chilo suppressalis epidermal protein gene LCP16/17 sequence through PCR amplification reaction, wherein the process comprises the following steps:
using the cDNA template obtained in the step (3), and using SEQ ID NO.3 as an upstream primer and SEQ ID NO.4 as a downstream primer to obtain an epidermal protein gene LCP16/17 full-length fragment through PCR amplification;
purifying the PCR product by using a MiniBEST Agarose Gel DNA Extraction Kit (Takara), connecting the purified product to a pEASY-T3 Cloning Kit (Transfer company), transferring the product into competent cells, recovering, coating a plate, carrying out bacteria selection detection, and sending a bacteria solution to a Progesteron (China) Limited company for sequencing to obtain a Chilo suppressalis epidermal protein gene LCP16/17 sequence; the nucleotide sequence of the striped rice borer epidermal protein gene LCP16/17 is 330bp, the nucleotide sequence is SEQ ID NO.1, and the amino acid sequence corresponding to the striped rice borer epidermal protein gene LCP16/17 is the sequence shown in SEQ ID NO. 2;
designing an upstream Primer SEQ ID NO.6 and a downstream Primer SEQ ID NO.7 containing a T7 promoter through Primer 5.0 software according to the nucleotide sequence of the known Chilo suppressalis epidermal protein gene LCP 16/17;
SEQ ID NO.6:TAATACGACTCACTATAGGGCAGCGAGTTCGACCAACAAC；
SEQ ID NO.7:TAATACGACTCACTATAGGGGCGTGGTATCCGGTCTCATC；
step (6), carrying out PCR amplification by using the cDNA template obtained in the step (3) and the synthesized upstream primer SEQ ID NO.6 and downstream primer SEQ ID NO.7 containing the T7 promoter sequence; obtaining a gene fragment with the length of 216 bp; after being purified by the Kit, the target dsRNA of the chilo suppressalis epidermal protein gene LCP16/17 is obtained by in vitro Transcription synthesis according to the specification of a TranscriptAId T7 High Yield Transcription Kit (Thermo Fisher Scientific) Kit; obtaining a target dsRNA of chilo suppressalis epidermal protein gene LCP16/17, wherein the nucleotide sequence of the target dsRNA is a sequence shown in SEQ ID NO. 5;
mixing the efficient dendritic polymerization nano-carrier with dsRNA to obtain an RNA preparation for inhibiting the boring behavior of chilo suppressalis;
in the RNA preparation, the charge ratio of the dsRNA to the nano-carrier is 1:1-1: 200.
The Efficient dendritic polymerization nano-carrier is synthesized according to the method of ' Non-viral method for Efficient Co-transfection Co-transfection ' (Efficient Co-transfection Non-viral Methodology) ' published by Jessica A. Kretzmann, Cameron W. Evans, Marck Norret, Pilar blancafefort, K. Swaminathan Iyer in 2018 on Methods in Molecular Biology 1767.
In the step (3), the preparation process of extracting the total RNA of the chilo suppressalis third-instar larvae and performing reverse transcription to form the cDNA template is as follows:
selecting healthy and consistent-size third-instar chilo suppressalis larvae, and putting the larvae into liquid nitrogen for freezing; total RNA was extracted according to Takara Trizol kit, and the extracted total RNA was reverse-transcribed into first-strand cDNA using M-MLV reverse transcriptase, thereby obtaining a cDNA template required for PCR reaction.
In the step (4), the obtained Chilo suppressalis epidermal protein gene LCP16/17 sequence is obtained by predicting after performing bioinformatics analysis on SEQ ID NO. 1; bioinformatics analysis shows that the chilo suppressalis epidermal protein gene LCP16/17 codes 110 amino acids, the predicted isoelectric point is 5.24, and the protein molecular weight is 27.0 kDa.
In the step (4), the PCR system is as follows: 2 Xfast Taq enzyme 12.5. mu.L, Forward Primer 1. mu.L, Reverse Primer 1. mu.L, cDNA 1. mu.L, ddH2O9.5 mu L, the total volume is 25 mu L;
the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, and circulating for 30 times; extending for 10min at 72 ℃, and storing the PCR product in a refrigerator at-20 ℃.
In the step (6), the PCR amplification system is as follows: 2 Xfast Taq enzyme 25. mu.L, Forward Primer 2. mu.L, Reverse Primer 2. mu.L, cDNA 1. mu.L, ddH2O20 mu L, and the total volume is 50 mu L;
the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extending for 10min at 72 ℃;
the PCR product was purified using the MiniBEST Agarose Gel DNA Extraction Kit (Takara), dsRNA (dsCsLCP 16/17) was synthesized by in vitro Transcription as described in the TranscriptAID T7 High Yield Transcription Kit (Thermo Fisher Scientific) and the dsRNA concentration was measured with a NanoPhotometer microspectrophotometer and stored in an ultra-low temperature refrigerator for later use.
An application of an RNA preparation for inhibiting the boring behavior of chilo suppressalis in preventing and controlling chilo suppressalis is characterized in that dsLCP16/17 and a nano carrier are mixed and delivered into chilo suppressalis bodies through drip body surface, and dsEGFP and a blank control group which do not target any gene of chilo suppressalis are used as negative controls of dsLCP 16/17;
selecting 45 healthy first-instar larvae with consistent size for experiment; 2.5 μ g dsLCP16/17 per treatment group, mixing dsLCP16/17 with nanocarriers in proportion, incubating for 15min at room temperature, dropping the mixture on the back of larvae, treating for 15min, treating once every 48h, treating three times continuously, setting three biological replicates, each biological replicate being 15 beetles; simultaneously, 90 head worms are selected to establish a dsRNA control group and a blank control group, the dosage of dsEGFP and the nano-carrier is the same as that of the treatment group, three biological repetitions are also set, and 15 head worms are repeated in each biological repetition; placing the treated chilo suppressalis in a constant-temperature incubator at 28 ℃ for feeding, wherein the incubator is illuminated: dark time =16 h: 8h, the temperature is 28 +/-1 ℃, the relative humidity is 70 +/-5%, and the treatment and control groups are fed with fresh artificial feed every day; observing the growth and development conditions of the chilo suppressalis larvae every day, and counting the condition that the chilo suppressalis larvae burrow into the feed;
extracting Chilo suppressalis total RNA of a treatment group and a control group 24h after interference, wherein the control group comprises a dsEGFP control group and a blank control group, synthesizing cDNA by a Takara reverse transcription kit, respectively detecting the relative expression quantity of a target gene CsLCP16/17 and a housekeeping gene EF1-a by adopting a Real-timePCR method, and calculating the silencing efficiency;
compared with a control group, the nano-carrier transdermal delivery dsLCP16/17 remarkably inhibits the expression amount of LCP16/17 gene; the dsRNA segment can remarkably silence the expression level of the target gene CsLCP16/17, is effective and can be used for follow-up research;
observing the growth condition of the chilo suppressalis after the intravenous drip treatment, counting the boring situation, and reducing the chilo suppressalis boring worm number ratio of the intravenous drip RNAi treatment group by 46.3 percent compared with the control group; compared with the EGFP dsRNA serving as a gene of a control group, the rate of the number of chilo suppressalis borers in the drop RNAi treatment group is reduced by 29 percent. Therefore, the RNA interference sequence and the drop RNAi method based on the nano-carrier can be applied to green prevention and control of Chilo suppressalis.
In the step (7), the charge ratio of the dsRNA to the nano-carrier is 1: 64;
when the feed is used, an RNA preparation interfering LCP16/17 genes is delivered into the bodies of the chilo suppressalis by dropping the body surface of the chilo suppressalis, the chilo suppressalis first-instar larvae are treated by dropping for 4 times continuously, the growth and development conditions of the chilo suppressalis larvae are observed once every 48 hours, and the situation that the chilo suppressalis larvae bore the feed is counted.
Compared with the prior art, the invention has the following beneficial effects:
according to the technical scheme, firstly, the chilo suppressalis epidermal protein gene LCP16/17 is cloned from chilo suppressalis, a primer is designed for the chilo suppressalis epidermal protein gene LCP16/17, dsRNA for interfering with the chilo suppressalis epidermal protein gene LCP16/17 is synthesized, a system for delivering the dsRNA by using a nano carrier is created, and the dsRNA is delivered into a worm body through dropping body surfaces to interfere with the chilo suppressalis epidermal protein gene LCP 16/17. The results show that: after the dsRNA is injected into the chilo suppressalis, the change of the boring behavior of the chilo suppressalis is found, the occurrence of the boring behavior is obviously reduced, and the dsRNA has an important effect on the prevention and the treatment of the chilo suppressalis. The invention provides a new method for molecular regulation of pests and also provides a theoretical basis for deeply understanding the growth and development mechanism of insects and creating a new biological pesticide preparation.
Drawings
FIG. 1 is a graph showing the silencing efficiency of Chilo suppressalis LCP16/17 gene after dropping dsRNA of LCP16/17 gene; wherein dsLCP16/17 represents the treatment group of dropping LCP16/17 gene dsRNA; dsEGFP represents a control group spotted with dsRNA for the EGFP gene; CK represents a blank control group;
FIG. 2 is a graph showing the change of dsRNA of drip LCP16/17 gene on boring behavior of Chilo suppressalis; wherein dsLCP16/17 represents the treatment group of dropping LCP16/17 gene dsRNA; dsEGFP represents a control group spotted with dsRNA for the EGFP gene; CK denotes blank control.
Detailed Description
The technical solution of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings, but the present invention is not limited thereto.
Example 1
Obtaining a full-length cDNA sequence of a chilo suppressalis epidermal protein gene LCP16/17 (CsLCP16/17) and analyzing an amino acid sequence of the cDNA sequence;
1. obtaining chilo suppressalis epidermal protein gene LCP16/17cDNA fragments;
according to a known insect CPR family protein sequence conserved region (motif), searching in a Chilo suppressalis transcriptome database by using a bioinformatics analysis method to obtain the LCP16/17 gene fragment.
2. Obtaining a chilo suppressalis epidermal protein gene LCP16/17cDNA full-length sequence;
1) designing primers required by PCR amplification;
based on the known LCP16/17 gene fragment, the upstream Primer SEQ ID NO.3 and the downstream Primer SEQ ID NO.4 were designed using Primer 5.0 software, which was synthesized by Populus organisms (China) Ltd.
2) Preparing a template required by PCR amplification;
selecting three-instar Chilo suppressalis larvae which grow healthily and are consistent in size, and putting the larvae into liquid nitrogen for freezing. Total RNA was extracted according to Takara Trizol kit, and the extracted total RNA was reverse-transcribed into first-strand cDNA using M-MLV reverse transcriptase, thereby obtaining a template required for PCR reaction.
3) PCR amplification reaction
The full-length fragment of the epidermal protein gene LCP16/17 is obtained by PCR amplification by using the template and the primer obtained in the previous step.
The PCR system is as follows: 2 Xfast Taq enzyme 12.5. mu.L, Forward Primer 1. mu.L, Reverse Primer 1. mu.L, cDNA 1. mu.L, ddH2O9.5. mu.L, total volume 25. mu.L.
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, and circulating for 30 times; extending for 10min at 72 ℃, and storing the PCR product in a refrigerator at-20 ℃.
Purifying the PCR product by using a MiniBEST Agarose Gel DNA Extraction Kit (Takara), connecting the purified product to a pEASY-T3 Cloning Kit (Transfer company), transferring the product into a competent cell, recovering, coating a plate, carrying out bacteria selection detection, sending a bacteria liquid to a Proctinathic organism (China) Limited company for sequencing to obtain the sequence of the chilo suppressalis epidermal protein gene LCP16/17 nucleotide sequence shown as SEQ ID NO. 1.
3. Analyzing the amino acid sequence of chilo suppressalis epidermal protein gene LCP 16/17;
the results of on-line software ExPASy analysis show that CsLCP16/17 encodes 110 amino acids, the predicted isoelectric point is 5.24, and the protein molecular weight is 27.0 kDa. The sequence with the amino acid sequence shown as SEQ ID NO.2 is obtained by sequencing.
Example 2
Synthesis of targeted dsRNA of Chilo suppressalis epidermal protein gene LCP 16/17:
1. designing a Chilo suppressalis epidermal protein gene LCP16/17 targeting dsRNA primer;
based on the sequence of Chilo suppressalis epidermal protein gene LCP16/17, the Primer 5.0 software is used for designing a targeting dsRNA Primer, and a T7 promoter is added at the 5' end of the Primer. The sequences of the primers are as follows:
SEQ ID NO.6：TAATACGACTCACTATAGGGCAGCGAGTTCGACCAACAAC
SEQ ID NO.7：TAATACGACTCACTATAGGGGCGTGGTATCCGGTCTCATC
2. synthesizing specific dsRNA of chilo suppressalis epidermal protein gene LCP 16/17;
using the cDNA obtained in the previous step as a template, PCR amplification was carried out using the above-synthesized upstream and downstream primers containing the T7 promoter sequence. Obtaining a gene fragment (SEQ ID NO. 5) with the length of 216 bp.
The PCR amplification system is as follows: 2 Xfast Taq enzyme 25. mu.L, Forward Primer 2. mu.L, Reverse Primer 2. mu.L, cDNA 1. mu.L, ddH2O20. mu.L, total volume 50. mu.L.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extension at 72 ℃ for 10 min.
The PCR product was purified using the MiniBEST Agarose Gel DNA Extraction Kit (Takara), dsRNA (dsCsLCP 16/17) was synthesized by in vitro Transcription as described in the TranscriptAID T7 High Yield Transcription Kit (Thermo Fisher Scientific) and the dsRNA concentration was measured with a NanoPhotometer microspectrophotometer and stored in an ultra-low temperature refrigerator for later use.
Example 3
In order to verify the interference effect of dsLCP16/17 on LCP16/17 genes, dsLCP16/17 prepared by the method is mixed with a nano carrier and delivered into chilo suppressalis bodies through drip body surfaces. The dsEGFP and blank control group, which did not target any gene of Chilo suppressalis, were used as negative controls for dsLCP 16/17.
45 healthy first instar larvae with consistent size are selected for experiment. 2.5 μ g dsLCP16/17 per treatment group, mixing dsLCP16/17 with nanocarriers in proportion, incubating for 15min at room temperature, dropping the mixture on the back of larvae, treating for 15min, treating once every 48h, treating three times continuously, setting three biological replicates, each biological replicate being 15 beetles; and simultaneously, 90 head worms are selected to establish a dsRNA control group and a blank control group, the dosage of the dsEGFP and the nano-carrier is the same as that of the treatment group, three biological repetitions are also set, and 15 head worms are repeated in each biological repetition. The treated chilo suppressalis is placed in a constant-temperature incubator at 28 ℃ for feeding (the light irradiation: dark time =16 h: 8h, the temperature is 28 +/-1 ℃, and the relative humidity is 70 +/-5%), and the treatment and control groups are fed with fresh artificial feed every day. And (4) observing the growth and development conditions of the chilo suppressalis larvae every day, and counting the condition of the chilo suppressalis larvae boring into the feed.
Extracting Chilo suppressalis total RNA of a treatment group and a control group (a dsEGFP control group and a blank control group) 24h after interference, synthesizing cDNA by a Takara reverse transcription kit, respectively detecting the relative expression quantity of a target gene (CsLCP16/17) and a housekeeping gene (EF1-a) by adopting a Real-timePCR method, and calculating the silencing efficiency.
As shown in FIG. 1, the nanocarrier transdermal delivery of dsLCP16/17 significantly inhibited the expression level of LCP16/17 gene compared to the control group. The dsRNA segment can remarkably silence the expression level of a target gene (CsLCP16/17), is effective and can be used for subsequent research.
Observing the growth condition of the chilo suppressalis after the intravenous drip treatment, and counting the diamond-eating condition, wherein as shown in figure 2, compared with the control group, the rate of the chilo suppressalis diamond-eating number of the intravenous drip RNAi treatment group is reduced by 46.3%; compared with the EGFP dsRNA serving as a gene of a control group, the rate of the number of chilo suppressalis borers in the drop RNAi treatment group is reduced by 29 percent. Therefore, the RNA interference sequence and the drop RNAi method based on the nano-carrier provided by the invention can be applied to green prevention and control of Chilo suppressalis.
Sequence listing
<110> Yangzhou university
<120> an RNA preparation for inhibiting Chilo suppressalis borer boring behavior
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 330
<212> DNA
<213> Chilo suppersalis
<400> 1
atgaaatcga tcgtattaat cgcccttttc gtggctgtcg ccgccgccct ccccgtggag 60
caggagcccg tgaagatcct tcgcagcgag ttcgaccaac aacccgaggg aggatacgcg 120
ttcagctttg agactgagga tggtgttgaa cggtctgaaa atggagaagt gaagcaggct 180
gttgatgagg aaaacaagcc ccatgacgtc gttgtagtac gtggctccta ctcctacacc 240
aaccctgacg gccagaagga gagcgtctct tactacgctg atgagaccgg ataccacgcg 300
gagggtgact ccatcccaaa ggtccaataa 330
<210> 2
<211> 109
<212> PRT
<213> Chilo suppersalis
<400> 2
Met Lys Ser Ile Val Leu Ile Ala Leu Phe Val Ala Val Ala Ala Ala
1 5 10 15
Leu Pro Val Glu Gln Glu Pro Val Lys Ile Leu Arg Ser Glu Phe Asp
20 25 30
Gln Gln Pro Glu Gly Gly Tyr Ala Phe Ser Phe Glu Thr Glu Asp Gly
35 40 45
Val Glu Arg Ser Glu Asn Gly Glu Val Lys Gln Ala Val Asp Glu Glu
50 55 60
Asn Lys Pro His Asp Val Val Val Val Arg Gly Ser Tyr Ser Tyr Thr
65 70 75 80
Asn Pro Asp Gly Gln Lys Glu Ser Val Ser Tyr Tyr Ala Asp Glu Thr
85 90 95
Gly Tyr His Ala Glu Gly Asp Ser Ile Pro Lys Val Gln
100 105
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaaatcga tcgtattaat 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttattggacc tttgggatgg 20
<210> 5
<211> 216
<212> DNA
<213> Chilo suppersalis
<400> 5
cagcgagttc gaccaacaac ccgagggagg atacgcgttc agctttgaga ctgaggatgg 60
tgttgaacgg tctgaaaatg gagaagtgaa gcaggctgtt gatgaggaaa acaagcccca 120
tgacgtcgtt gtagtacgtg gctcctactc ctacaccaac cctgacggcc agaaggagag 180
cgtctcttac tacgctgatg agaccggata ccacgc 216
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taatacgact cactataggg cagcgagttc gaccaacaac 40
<210> 7
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
taatacgact cactataggg gcgtggtatc cggtctcatc 40
Claims (7)
1. An RNA preparation for inhibiting the boring behavior of chilo suppressalis, which is characterized by comprising the following steps:
searching in a chilo suppressalis transcriptome database by using a bioinformatics analysis method according to a known insect CPR family protein sequence conserved region motif to obtain an LCP16/17 gene fragment;
step (2), designing an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4 according to the known LCP16/17 gene fragment by using primer premier 5.0 software;
SEQ ID NO.3:ATGAAATCGATCGTATTAAT；
SEQ ID NO.4:TTATTGGACCTTTGGGATGG；
extracting total RNA of chilo suppressalis third-instar larvae and performing reverse transcription to obtain a cDNA template;
step (4), obtaining a Chilo suppressalis epidermal protein gene LCP16/17 sequence through PCR amplification reaction, wherein the process comprises the following steps:
using the cDNA template obtained in the step (3), and using SEQ ID NO.3 as an upstream primer and SEQ ID NO.4 as a downstream primer to obtain an epidermal protein gene LCP16/17 full-length fragment through PCR amplification;
purifying the PCR product by using a MiniBEST Agarose Gel DNA Extraction Kit (Takara), connecting the purified product to a pEASY-T3 Cloning Kit (Transfer company), transferring the product into competent cells, recovering, coating a plate, carrying out bacteria selection detection, and then sequencing bacteria liquid to obtain a Chilo suppressalis epidermal protein gene LCP16/17 sequence; the nucleotide sequence of the striped rice borer epidermal protein gene LCP16/17 is 330bp, the nucleotide sequence is SEQ ID NO.1, and the amino acid sequence corresponding to the striped rice borer epidermal protein gene LCP16/17 is the sequence shown in SEQ ID NO. 2;
designing an upstream Primer SEQ ID NO.6 and a downstream Primer SEQ ID NO.7 containing a T7 promoter through Primer 5.0 software according to the nucleotide sequence of the known Chilo suppressalis epidermal protein gene LCP 16/17;
SEQ ID NO.6:TAATACGACTCACTATAGGGCAGCGAGTTCGACCAACAAC；
SEQ ID NO.7:TAATACGACTCACTATAGGGGCGTGGTATCCGGTCTCATC；
step (6), carrying out PCR amplification by using the cDNA template obtained in the step (3) and the synthesized upstream primer SEQ ID NO.6 and downstream primer SEQ ID NO.7 containing the T7 promoter sequence; obtaining a gene fragment with the length of 216 bp; after being purified by the Kit, the target dsRNA of the chilo suppressalis epidermal protein gene LCP16/17 is obtained by in vitro Transcription synthesis according to the specification of a TranscriptAId T7 High Yield Transcription Kit (Thermo Fisher Scientific) Kit; obtaining a target dsRNA of chilo suppressalis epidermal protein gene LCP16/17, wherein the nucleotide sequence of the target dsRNA is a sequence shown in SEQ ID NO. 5;
mixing the efficient dendritic polymerization nano-carrier with dsRNA to obtain an RNA preparation for inhibiting the boring behavior of chilo suppressalis;
in the RNA preparation, the charge ratio of the dsRNA to the nano-carrier is 1:1-1: 200.
2. The RNA preparation for inhibiting Chilo suppressalis borer boring behavior according to claim 1, wherein in the step (3), the total RNA of Chilo suppressalis third instar larvae is extracted and reverse transcribed into cDNA template, and the preparation process comprises the following steps:
selecting healthy and consistent-size third-instar chilo suppressalis larvae, and putting the larvae into liquid nitrogen for freezing; total RNA was extracted according to Takara Trizol kit, and the extracted total RNA was reverse-transcribed into first-strand cDNA using M-MLV reverse transcriptase, thereby obtaining a cDNA template required for PCR reaction.
3. The RNA preparation for inhibiting chilo suppressalis borer boring behavior according to claim 1, wherein the obtained chilo suppressalis epidermal protein gene LCP16/17 sequence in the step (4) is predicted after bioinformatics analysis of SEQ ID No. 1; bioinformatics analysis shows that the chilo suppressalis epidermal protein gene LCP16/17 codes 109 amino acids, the predicted isoelectric point is 5.24, and the protein molecular weight is 27.0 kDa.
4. The RNA preparation for inhibiting Chilo suppressalis borer boring behavior according to claim 1, wherein in the step (4), the PCR system is as follows: 2 Xfast Taq enzyme 12.5. mu.L, Forward Primer 1. mu.L, Reverse Primer 1. mu.L, cDNA 1. mu.L, ddH2O9.5 mu L, the total volume is 25 mu L;
the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, and circulating for 30 times; extending for 10min at 72 ℃, and storing the PCR product in a refrigerator at-20 ℃.
5. The RNA preparation for inhibiting Chilo suppressalis borer boring behavior according to claim 1, wherein in the step (6), the PCR amplification system comprises: 2 Xfast Taq enzyme 25. mu.L, Forward Primer 2. mu.L, Reverse Primer 2. mu.L, cDNA 1. mu.L, ddH2O20 mu L, and the total volume is 50 mu L;
the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extending for 10min at 72 ℃;
the PCR product was purified using the MiniBEST Agarose Gel DNA Extraction Kit (Takara), dsRNA (dsCsLCP 16/17) was synthesized by in vitro Transcription as described in the TranscriptAID T7 High Yield Transcription Kit (Thermo Fisher Scientific) and the dsRNA concentration was measured with a NanoPhotometer microspectrophotometer and stored in an ultra-low temperature refrigerator for later use.
6. The use of the RNA preparation for inhibiting the boring behavior of the chilo suppressalis as claimed in any one of claims 1 to 5 for preventing and controlling the chilo suppressalis, wherein dsLCP16/17 is mixed with a nano-carrier and delivered into the chilo suppressalis body surface by dropping, and dsEGFP which does not target any gene of the chilo suppressalis and a blank control group are used as a negative control of dsLCP 16/17;
selecting 45 healthy first-instar larvae with consistent size for experiment; 2.5 μ g dsLCP16/17 per treatment group, mixing dsLCP16/17 with nanocarriers in proportion, incubating for 15min at room temperature, dropping the mixture on the back of larvae, treating for 15min, treating once every 48h, treating three times continuously, setting three biological replicates, each biological replicate being 15 beetles; simultaneously, 90 head worms are selected to establish a dsRNA control group and a blank control group, the dosage of dsEGFP and the nano-carrier is the same as that of the treatment group, three biological repetitions are also set, and 15 head worms are repeated in each biological repetition; placing the treated chilo suppressalis in a constant-temperature incubator at 28 ℃ for feeding, wherein the incubator is illuminated: dark time =16 h: 8h, the temperature is 28 +/-1 ℃, the relative humidity is 70 +/-5%, and the treatment and control groups are fed with fresh artificial feed every day; observing the growth and development conditions of the chilo suppressalis larvae every day, and counting the condition that the chilo suppressalis larvae burrow into the feed;
extracting Chilo suppressalis total RNA of a treatment group and a control group 24h after interference, wherein the control group comprises a dsEGFP control group and a blank control group, synthesizing cDNA by a Takara reverse transcription kit, respectively detecting the relative expression quantity of a target gene CsLCP16/17 and a housekeeping gene EF1-a by adopting a Real-timePCR method, and calculating the silencing efficiency;
compared with a control group, the nano-carrier transdermal delivery dsLCP16/17 remarkably inhibits the expression amount of LCP16/17 gene; the dsRNA segment can remarkably silence the expression level of the target gene CsLCP16/17, is effective and can be used for follow-up research;
observing the growth condition of the chilo suppressalis after the intravenous drip treatment, counting the boring situation, and reducing the chilo suppressalis boring worm number ratio of the intravenous drip RNAi treatment group by 46.3 percent compared with the control group; compared with EGFP dsRNA of a gene of a control group, the rate of the number of chilo suppressalis borers in the drop RNAi treatment group is reduced by 29 percent; therefore, the RNA interference sequence and the drop RNAi method based on the nano-carrier can be applied to green prevention and control of Chilo suppressalis.
7. The RNA preparation for inhibiting Chilo suppressalis borer boring behavior according to claim 1, wherein in the step (7), the charge ratio of dsRNA to nano-carrier is 1: 64;
when the feed is used, an RNA preparation interfering LCP16/17 genes is delivered into the bodies of the chilo suppressalis by dropping the body surface of the chilo suppressalis, the chilo suppressalis first-instar larvae are treated by dropping for 4 times continuously, the growth and development conditions of the chilo suppressalis larvae are observed once every 48 hours, and the situation that the chilo suppressalis larvae bore the feed is counted.
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| CN114854750B (en) * | 2022-05-12 | 2024-04-09 | 扬州大学 | Interference RNA (ribonucleic acid) for inhibiting development of head shells of Chilo suppressalis larvae, kit and use method |
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Application publication date: 20211214 |