Spodoptera cunea Rop gene dsRNA as well as bacterial expression solution and application thereof
阅读说明：本技术 美国白蛾Rop基因dsRNA及其细菌表达液和应用 (Spodoptera cunea Rop gene dsRNA as well as bacterial expression solution and application thereof ) 是由 张真 张珣 张苏芳 樊智智 孔祥波 刘福 方加兴 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种美国白蛾Rop基因dsRNA及其细菌表达液和应用,该Rop基因dsRNA片段的核苷酸序列如SEQ ID NO.2所示。本发明采用重组质粒在大肠杆菌HT115中表达大量所需的外源目的dsRNA,持续饲喂IPTG诱导表达靶标dsRNA的大肠杆菌HT115菌液后,对美国白蛾具有高致死能力,且对其生长发育有显著的抑制作用,采用本发明的细菌表达液防治美国白蛾的方法可实施性强、操作方便、有效性和灵敏性好、杀虫效率高,且具有环境友好等诸多优点,有很好的应用前景。(The invention discloses a fall webworm Rop gene dsRNA, bacterial expression liquid thereof and application thereof, wherein the nucleotide sequence of the Rop gene dsRNA fragment is shown as SEQ ID NO. 2. The method for preventing and treating fall webworm by using the bacterial expression liquid has the advantages of strong feasibility, convenient operation, good effectiveness and sensitivity, high insecticidal efficiency, environmental friendliness and the like, and has good application prospect.)
1. The fall webworm Rop gene dsRNA is characterized in that the nucleotide sequence of the dsRNA segment is shown as SEQ ID NO. 2.
2. A bacterial expression fluid expressing the dsRNA of claim 1, prepared by a method comprising:
(1) performing PCR amplification by using a dsHcRop primer by using the hyphal moth cDNA as a template to obtain the dsRNA segment, wherein the sequences of the dsHcRop primer are shown as SEQ ID NO.10 and 11;
(2) connecting the dsRNA segment to a linear plasmid to construct a recombinant vector;
(3) introducing a recombinant expression vector containing the dsRNA segment into a bacterium competent cell for culture;
(4) dsRNA production is induced by IPTG, and bacterial liquid is cultured and collected.
3. The bacterial expression fluid of claim 2, wherein the plasmid is linearized L4440 and/or the bacterium is e.
4. Use of the dsRNA of claim 1 or the bacterial expression fluid of claim 2 for controlling fall webworm.
5. The use of claim 4, wherein the control of fall webworm is achieved by feeding or spraying a bacterial expression solution expressing Rop gene dsRNA.
6. The use of claim 5, wherein the control of fall webworm is achieved by feeding artificial feed mixed with bacterial expression solution expressing Rop gene dsRNA.
7. The use of claim 6, wherein the prepared artificial feed is cut into 0.5X0.5X0.5cm pieces per 30g, and sprayedThe concentration of dsRNA expressing the Rop gene is 1075mL of CFU/mL HT115 bacterial liquid is dried for 1h at normal temperature and fed.
8. The application of claim 5, wherein the control of fall webworm is realized by directly spraying a bacterial expression solution for expressing Rop gene dsRNA onto a plant.
9. The use of claim 8, wherein the bacterial expression fluid is Escherichia coli HT115 bacterial fluid.
Technical Field
The application relates to the technical field of molecular biology, in particular to a fall webworm Rop gene dsRNA, bacterial expression liquid thereof and application thereof.
Background
The fall webworm (Hyphantia cunea) belongs to the Lepidoptera lampwick family, is extremely dangerous external invasive forest pests in China, has wide host plants, can eat almost all kinds of cultivated trees, flowers and crops, and has large food consumption and strong adaptability because the larvae eat leaves in a colony form. The adult reproduction ability is strong, and the average egg laying of each female insect is 800-900, and the maximum egg laying can reach more than 2000. Meanwhile, the adult diffusion capacity is very strong, and the male insects can fly more than 7 kilometers and at most 23 kilometers in 12 hours. The characteristics lead the fall webworms to be extremely easy to outbreak and cause disasters, the fall webworms have caused huge economic loss and serious ecological threat to the ecological system of agriculture and forestry in China since being discovered for the first time in China in 1979, once the fall webworms invade a new suitable place, the fall webworms are difficult to be thoroughly eliminated, and the ecological safety of China is seriously threatened.
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by the entry of double-stranded RNA (dsRNA) into cells, which effectively leads to pest death by silencing specific lethal genes. Although the RNAi technology has a wide application prospect as a new means for pest control, numerous studies find that the RNAi efficiency of different insects is obviously different, generally, the RNAi efficiency of insects such as Coleoptera, Orthoptera and the like is high, but the RNAi efficiency of Lepidoptera insects is very low (Wang, et al, "Variation in RNAi efficiency amplifying insects is effective to dsRNA deletion in vivo". Insect Biochemistry and Molecular Biology,77,1-9,2016; Terenius, et al, "RNA interference in Lepidoptera: An overview of social and environmental characteristics and for experimental purposes". Journal of Insect physiology.57(2), 245,2011) and the application of RNAi technology in Lepidoptera pest control seriously affects the application of RNAi technology in Lepidoptera pest control. Among the many factors that affect RNAi efficiency, one of the known factors is the stability of dsRNA, and maintaining the effectiveness of RNAi must avoid degradation of dsRNA, such as double-stranded RNA degrading enzymes (dsRNases), a class of dsRNA specific degrading enzymes, whose Expression has been currently detected in different tissues of lepidopteran insects such as Bombyx mori, Spodoptera litura (Liu, et al), "Bombyx mobile DNA/RNA non-specific genes: Expression of infectious in culture cells, subellicular localization and functional analysis". J. of infection physiology.58 (1168), 1166. 1176, 2012; Penga, et al, "Identification and characterization of bacteria," biological and mutation of bacteria, "emission and mutation of RNA, 95,2020. It was found that the activity of dsRNases in lepidopteran insects is higher than that of other groups of insects (Kun Yan Zhu and Subba Red Palli, "Mechanisms, applications, and changes of infection RNA interference". Annual Review of Entomology,65,293 and 311,2020), and that degradation of dsRNases may be a major factor limiting the RNAi efficiency of lepidopteran insects. Therefore, RNAi is difficult to realize in lepidoptera insects, and efficient RNAi target genes are rarely reported in the control of lepidoptera pests.
As an important quarantine pest, the control of the fall webworm is mainly chemical control at present, so that the negative influence on the ecological environment is caused, biological control measures such as parasitic natural enemies, sex attractants and the like are also used, but the problems of low efficiency, high cost and the like exist totally, and the report of the RNAi target gene with high efficient insecticidal activity of the fall webworm does not exist at home and abroad at present. Although patent application CN 111944824A discloses application of a tachykinin receptor gene and dsRNA of the fall webworm in the fall webworm control, the dsRNA is ingested by an injection method in the application and cannot be applied on a large scale in the actual forest control, and the tachykinin receptor gene and the dsRNA in the application can only reduce the food intake and hunger-resistant capability of the fall webworm larva, weaken the life of the fall webworm, and achieve no lethal effect, and have general control effect. Yan Xiaoping (Biochemical characterization and functional research of the hyphantria cunea chitin deacetylase HcCDAs, university of Hebei agriculture (academic paper), 2018.) can achieve more than 80% of larva mortality rate by injecting dsRNA of the hyphantria cunea chitin deacetylase gene into 5-instar larvae of hyphantria cunea, but the injection dose is 10ug, and the injection dose is too high to achieve in actual pest control. Wangyue ('RNA interference-based fall webworm gene function research and transcriptome analysis', China forestry science research institute (academic paper), 2018) constructs an HT115 strain expression system of fall webworm chitinase gene dsRNA, but after the recombinant bacteria are fed, the deformed phenotype and the change of pupation rate and death rate are not generated, only the weight of larvae is obviously reduced, and the prevention and treatment effect is not ideal.
The Rop protein is involved in intracellular vesicle trafficking, and is crucial for vesicle trafficking and membrane fusion if the Rop of flies is a homologue of the yeast Sec.1 protein (Fujita, Y.et., Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C-Its immunization in regulating the interaction of Munc-18/n-Sec1/rbSec1 with synthesis ". Journal of Biological Chemistry,271(13):7265-7268, 1996). The Rop gene has an important regulation and control function for maintaining the cell activity, and may indirectly influence the physiological processes of development, growth, reproduction, metabolism and the like of insects. At present, no report is found about the prevention and control of the major quarantine pest hyphantria cunea by inhibiting the transcription level of the Rop gene of the insect.
Disclosure of Invention
In order to overcome the defects and the defects of the existing lepidoptera pest fall webworm control technology, the invention provides fall webworm Rop gene dsRNA, and the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 2.
The invention also provides a bacterial expression solution for expressing the dsRNA, and the preparation method of the bacterial expression solution comprises the following steps:
(1) performing PCR amplification by using a dsHcRop primer by using the hyphal moth cDNA as a template to obtain the dsRNA segment, wherein the sequences of the dsHcRop primer are shown as SEQ ID NO.10 and 11;
(2) connecting the dsRNA segment to a linear plasmid to construct a recombinant vector;
(3) introducing a recombinant expression vector containing the dsRNA segment into a bacterium competent cell for culture;
(4) dsRNA production is induced by IPTG, and bacterial liquid is cultured and collected.
Further, the plasmid is linearized L4440, and the bacterium is Escherichia coli HT 115.
The invention also provides application of the dsRNA or the bacterial expression solution in preventing and treating fall webworm.
Further, the control of the fall webworm is realized by feeding or spraying bacterial expression liquid for expressing dsRNA of Rop gene.
The control of the fall webworm is realized by feeding the artificial feed mixed with the bacterial expression solution for expressing the Rop gene dsRNA, and the control method specifically comprises the following steps: cutting the prepared artificial feed into blocks of 0.5x0.5x0.5cm size per 30g, and spraying dsRNA expressing Rop gene at a concentration of 1075mL of CFU/mL HT115 bacterial liquid is dried for 1h at normal temperature and fed.
Or directly spraying a bacterial expression solution for expressing the Rop gene dsRNA on the plant to realize the control of the fall webworm, wherein the bacterial expression solution is an escherichia coli HT115 bacterial solution.
The beneficial effects of the invention include:
the invention obtains a fall webworm RNAi high-efficiency lethal target gene Rop gene by screening, develops a technology capable of efficiently preventing and controlling the fall webworm based on the Rop gene, adopts recombinant plasmids to express a large amount of needed exogenous target dsRNA in escherichia coli HT115, has high lethal capability to the fall webworm after continuously feeding escherichia coli HT115 bacterial liquid of IPTG inducible expression target dsRNA, and has obvious inhibition effect on the growth and development of the fall webworm. Compared with the in vitro transcription and chemical synthesis method of the kit adopted in the prior art, the invention can greatly reduce the experiment cost.
Drawings
FIG. 1 is an electrophoresis diagram of Rop gene of fall webworm;
FIG. 2 is the larval mortality rate for normal growing larvae (control), dsGFP-injected control group (dsGFP) and dsHcRop-injected treatment group (dsHcRop) 8d to 13d after the injection of the hyphal moth dsHcRop;
FIG. 3 is the phenotypic differences at pupation stage 13d after injection of the hyphantria cunea dsHcRop, normal grown larvae (control), dsGFP-injected control group (dsGFP) and dsHcRop-injected treatment group (dsHcRop);
FIG. 4 is a graph showing the relative expression amounts of Rop genes in larvae of normal grown larvae (control), control group injected with dsGFP (dsGFP) and treatment group injected with dsHcRop (dsHcRop) 48h after injection of dsRNA of Rop gene of fall webworm;
FIG. 5 shows mortality and pupation rates of larvae from 12d to 18d after feeding E.coli cell culture expressing the hyphal moth dsHcRop, control (control) and control (dsHcRop) treated with the dsHcRop cell culture.
Detailed Description
The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 full-Length cloning of Rop Gene of Spodoptera cunea
(1) The hyphantria cunea larvae are taken, and the total RNA of the hyphantria cunea is extracted by a TRIzol method (Trizol Plus reagent, Ambion, Austin, TX, USA).
(2) Using the Reverse Transcription kit GoScriptTM Reverse Transcription System kit (Promega, Ma)dison, WI, USA) was synthesized for the first strand cDNA, the reverse transcription system was: total RNA 1. mu.g, oligo (dT)15Primer(500μg/ml)0.5μL，GoScriptTM 5X Reaction Buffer 4μL，MgCl2(25mM)1μL，Random Primers(500μg/ml)0.5μL，PCR Nucleotide Mix 1μL，RecombinantRibonucleae Inhibitor 0.4. mu.L, GoScriptTM Reverse Transcriptase 1. mu.L, Nuclear-Free Water make-up 20. mu.L. The reaction conditions are 42 deg.C, 15min, 70 deg.C, 15 min.
(3) Primers were designed on both sides of the gene coding region sequence according to the fall webworm larva transcriptome sequence. Primers were designed using Primer5 software to give forward and reverse primers.
A forward primer: ATGTATAATCCTGCTCTTGCC (SEQ ID NO.4 in the sequence Listing)
Reverse primer: TTAGGCGGTTAGAGAGCTCAA (SEQ ID NO.5 in the sequence Listing)
And (3) taking the first strand of the cDNA obtained by reverse transcription as a template, and amplifying by PCR to obtain a target product fragment. The PCR reaction system is as follows: cDNA 2. mu.L, Buffer (Mg)2+Plus)5μL，dNTP Mixture 8μL，Forward Primer 2μL，Reverse Primer2μL，Max DNA Polymerase 0.5. mu.L, plus ddH2O to 100. mu.L. The reaction conditions are 94 ℃ for 1 min; 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃.
(4) The PCR products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 1. And (4) purifying and recovering the target product by using a DNA purification kit (Tiangen). After recovery, the product was ligated to pEASY-Blunt vector (TransGen, Beijing, China), transferred to DH5 alpha competent cells (TransGen, Beijing, China), spread on Amp-resistant selection medium, positive single colonies were picked up in Amp-resistant liquid LB medium and sent to the company (Sangon Biotech, Co., Ltd., Beijing, China) for sequencing.
(5) And comparing the sequencing result with an NCBI database to obtain a sequence fragment of the Rop gene of the fall webworm, wherein the specific nucleotide sequence is shown as SEQ ID NO.1 in the sequence table. The sequencing result verifies that the correct plasmid is the plasmid with the sequence of SEQ ID NO. 1.
Example 2 Synthesis of dsRNA of Rop Gene of fall webworm
(1) The plasmid (plasmid having the sequence of SEQ ID NO. 1) whose sequencing result confirmed the correctness was continuously subjected to PCR amplification using dsRNA primer having the sequence of T7 promoter, and the amplification method and system were referred to the above step (3) of example 1. The amplification product is recovered and purified according to the method of recovering and purifying the amplification product described in step (4) of example 1.
The PCR amplification primers are as follows:
taatacgactcactataggGCACGCCCAACTCGACCCCT (SEQ ID NO.6 in the sequence Listing)
taatacgactcactataggCGCCGCCAACTACGAAAACA (SEQ ID NO.7 in the sequence Listing)
(2) And (2) purifying and recovering the PCR amplification product obtained in the step (1) to obtain a dsRNA in-vitro transcription template.
In vitro synthesis of dsRNA T7 RiboMAXTM Express RNAi systems kit (Promega, Madison, WI, USA) was used in the reaction System: RiboMAXTM Express T72X Buffer 10. mu.L, linear DNA template about 1. mu.g, enzyme mix, T7 Express 2. mu.L, make up nuclease free water to 20. mu.L. Gently mixed and incubated at 37 ℃ for 3 h.
(3) Annealing of double-stranded RNA was achieved by mixing equal volumes of complementary RNA reaction solutions, incubating at 70 ℃ for 10 minutes, and slowly cooling to room temperature (about 20 min). mu.L of RNase was added to 199. mu.L of nuclease-free water to dilute the attached RNase solution (1: 200). mu.L of freshly diluted RNase solution and 1. mu.L of RQ1 RNase-Free DNase were added, respectively, and incubated at 37 ℃ for 30 minutes to remove all remaining single-stranded RNA and DNA template, leaving only double-stranded RNA.
(4) 0.1 volume of 3M sodium acetate (pH 5.2) and 1 volume of isopropanol were added, mixed well and placed on ice for 5 minutes. 12000rpm, 4 ℃ centrifugal 10 minutes, the centrifugal tube bottom visible white precipitation. The supernatant was discarded, the pellet was washed with 0.5mL of 70% cold ethanol, air-dried at room temperature, and then dissolved in 4 volumes of nuclease-free water to obtain purified dsRNA, which was stored at-80 ℃ for further use.
(5) 1 microliter of synthesized dsRNA is taken to carry out a 1% agarose gel electrophoresis experiment, the voltage condition of electrophoresis is 120 volts, the time is 15 minutes, the used buffer solution needs DEPC processing water to be configured, the sample application needs to use an RNAase-free gun head, a single clear band (the specific nucleotide sequence of the band is shown as SEQ ID NO.2 in a sequence table) is arranged at about 350bp of dsHcRop of the dsRNA segment, and the band can be used for subsequent experiments.
Example 3 growth inhibition of larvae by dsHcRop injection
(1) Selecting 4-year-old larvae of fall webworms with normal growth, consistent development and the same size, starving for 12h, placing 4-5 larvae in a culture dish before injection, performing cold anesthesia in a refrigerator at-20 ℃ for 2-3min, taking out, placing on an ice tray, and injecting 6 mu g of dsHcRop obtained in example 2 to the abdominal internode membrane of the larvae by using a micro-injector before the larvae do not return to autonomous movement. Injecting under a stereoscope, observing whether the injected liquid completely enters the polypide and has no leakage phenomenon, placing the larvae which are successfully injected and do not cause obvious mechanical damage in a new culture dish for self recovery, taking out the larvae which die due to non-experimental factors after 2h, continuously feeding the rest larvae according to normal conditions, and performing subsequent observation and sampling treatment. The experiment was performed with 2 control groups, i.e., normal growing larvae (control) and larvae injected with exogenous dsGFP fragment (exogenous dsGFP fragment is an exogenous fragment amplified from pGFP plasmid, and used as a control, size 678bp, nucleotide sequence of dsGFP fragment is shown in SEQ ID NO. 3) (dsGFP). Each treatment was injected with 30 larvae, one biological replicate for 10, and larval mortality was investigated starting 8 days after injection.
(2) The experimental results are shown in fig. 2, and there was no difference in the mortality of normal grown larvae (control) and dsGFP-injected larvae, but from 10d after treatment, the mortality of dsHcRop-injected larvae was significantly higher than that of the control group, which was 2.3 times and 1.8 times that of normal grown larvae and dsGFP-injected larvae, respectively. Pupation stage hyphantria cunea phenotype as shown in fig. 3, both normal growing (control) and dsGFP injected larvae pupate normally, but dsHcRop injected larvae develop malformed pupae.
Example 4 injection of dsHcRop inhibits Rop Gene expression in Spodoptera cunea
(1) Samples were taken 48h after dsHcRop injection and 3 larvae (3 biological replicates) were removed from each treatment group. RNA of the fall webworm was collected by extraction and then reverse-transcribed into cDNA in the same manner as in steps (1) and (2) of example 1.
(2) RT-qPCR primers are designed by using Primer5 software according to the sequence of SEQ ID NO.1, and the relative expression quantity of the Rop gene is detected. qPCR assays were performed using the SuperReal PreMix Plus (SYBRGreen) kit (tiangen). The reaction system was (20 μ L): 2 XSuperReal PreMix Plus 10. mu.L, forward primer (10. mu.M) 0.6. mu.L, reverse primer (10. mu.M) 0.6. mu.L, cDNA template 1. mu.L, nucleic-Free Water make-up 20. mu.L. The reaction conditions were 95 ℃ for 3min, 95 ℃ for 30s, 60 ℃ for 30s, 40 cycles, 3 technical replicates per sample, and the reaction was carried out in a Bio-Rad (CFX96 Touch) instrument.
The RT-qPCR primer is as follows:
f: GGAACAGATGCTGAAGGCGAGA (SEQ ID NO.8 in the sequence Listing)
R: CAGATTCTCCTCCGAAATGCCG (SEQ ID NO.9 in the sequence Listing)
(3) The experimental result is shown in fig. 4, after 48h of dsRNA injection, the exogenous dsGFP injection treatment has no significant difference from the normally bred larvae, and the expression of the Rop gene in the larvae injected with dsHcRop is significantly lower than that of the control group, which indicates that the dsHcRop injection can cause strong RNAi effect in the fall webworm, so that the expression level of the Rop gene in the fall webworm is obviously reduced, and the death or development of the fall webworm larvae is inhibited.
Example 5 preparation of bacterial expression bacterial solution expressing dsRNA expressing Rop Gene of fall webworm
(1) Two sites were selected on the L4440 plasmid (Addgene, Inc.), Bgl II (AGATCT) and PstI (CTGCAG), and PCR amplification was performed using the hypha moth cDNA as a template and dsHcRop primers with the corresponding cleavage sites and protected bases, and the amplification method and system were as described in example 1 (3). The amplification product is recovered and purified according to the method of recovering and purifying the amplification product described in step (4) of example 1.
The dsHcRop primers are:
GAAGATCTTCGCACGCCCAACTCGACCCCT (SEQ ID NO.10 in the sequence Listing)
AACTGCAGAACCAATGCATTGGCGCCGCCAACTACGAAAACA (SEQ ID NO.11 in the sequence Listing)
(2) The L4440 vector is linearized by BglII and PstI (Takara) according to the sequence of the two enzyme cutting sites, the reaction system of enzyme cutting is described in the specification, and after the enzyme cutting reaction is finished, the linearized L4440 vector is recovered by using a DNA purification recovery kit (Tiangen). Recombinant vectors were constructed by ligating purified dsHcRop fragments with linearized L4440 vector using T4 DNA ligase (TransGenBiotech) overnight at 4 ℃. Then, the recombinant expression vector containing dsHcRop is introduced into HT115 competent cells, placed on ice for 30min, then heat shocked at 42 ℃ for 1min, kept still on ice for 2min, then 500 microliter of LB liquid culture medium without ampicillin is added, and cultured for 1h at 37 ℃ and 200rpm, and then LB plate containing ampicillin and tetracycline is used for overnight culture to verify positive cloning, and bacterial expression bacteria liquid successfully expressing the hypha of the Rop gene of the fall webworm is obtained. HT 115-expressing strain expressing the recombinant vector was shake-cultured overnight at 1:100 in LB liquid medium containing ampicillin (100. mu.g/mL) and tetracycline (10. mu.g/mL), and shake-cultured at 37 ℃ for 3.5 hours to OD600Reaching 0.4-0.5), adding IPTG (final concentration of 1mM) to induce dsRNA to generate, and continuously culturing for 5h under the same condition to collect bacterial liquid for later use. RNA was extracted by the TRIzol method (Trizol Plus reagent, USA), and 1% agarose gel electrophoresis was used to verify whether dsRNA was successfully induced.
Example 6 control Effect of feeding dsHcRop-expressing bacterial solutions on fall webworm
(1) Cutting the prepared artificial feeding feed into blocks with the size of about 0.5x0.5x0.5cm per 30g, and spraying 5mL (the concentration is about 10) of HT115 bacterial liquid for successfully expressing dsHcRop7CFU/mL), the bacterial liquid preparation method is the same as example 5, and the larvae are fed after being air-dried for 1 hour at room temperature.
(2) Selecting 3-instar larvae of fall webworm with normal growth, consistent growth and same size, adding the above feed for normal feeding, and using HT115 bacterial liquid transferred into L4440 empty vector with same concentration as control. Each 90 larvae (30 were one biological replicate, 3 biological replicates) were treated and fed until larvae pupate. Larval mortality and pupation rates were investigated 12d after treatment.
(3) The experimental result is shown in fig. 5, after the HT115 bacterial liquid for expressing dsHcRop is continuously fed for 14d, the death rate of the fall webworm larvae is 65% which is 1.6 times that of the control group, and the pupation rate of the 18d larvae is 35% which is 0.6 times that of the control group, which indicates that the HT115 bacterial liquid for expressing dsHcRop can effectively inhibit the growth and development of the fall webworm larvae.
Sequence listing
<110> institute for forest ecological environment and protection of China forestry science research institute
<120> hypha cunea moth Rop gene dsRNA, bacterial expression solution and application thereof
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ggattcgact gtgtatctcc attactgcat gaactgactc ttcaagctat ggcatatgat 300
cttctgccta ttgagaatga tgtttataag tatgatgttt ccggaaacat caaagaagtc 360
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tccacatctg taacaaagaa cttgaagaaa tttacagaat ccaagcgcat gggtggaggc 480
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gaactatcca aatacgctac acatctgcgc ttagccgagg attgtatgaa atcctaccaa 600
ggctatgtgg acaagctgtg caaggtggaa caggatttgg caatgggaac agatgctgaa 660
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gtgaacttca acaagatgcg catcattgcg ttgtacatca tgtccaagaa cggcatttcg 780
gaggagaatc tgaacaaact tgtaacgcac gcccaactcg acccctcaga caagcagaca 840
ctgttgaacc ttgccaacct cggtcttaac gtcgttattg atggtaatag gaagaagcaa 900
taccaaatca caaggaagga aagaattact gaacaaacct accaaatgtc cagatggacg 960
ccagttatca aggatattat ggaagatgcc attgaagata aactggatca acgtcattat 1020
ccgttcctgg ctggtcgcgc gcaaacgtca ggataccaag cacctactag cgtacgctac 1080
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ttcgtagttg gcggcgtatg cttctctgaa attcgctgcg cctacgaagt tactgctgca 1200
ctcaagaact gggaggtcat tgttggctca tcacacatac tgacgccaga gaacttcctc 1260
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tactgaacaa acctaccaaa tgtccagatg gacgccagtt atcaaggata ttatggaaga 180
tgccattgaa gataaactgg atcaacgtca ttatccgttc ctggctggtc gcgcgcaaac 240
gtcaggatac caagcaccta ctagcgtacg ctacggacac tggcataagg ataaggccca 300
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ttaggcggtt agagagctca a 21
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
taatacgact cactataggg cacgcccaac tcgacccct 39
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
taatacgact cactataggc gccgccaact acgaaaaca 39
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggaacagatg ctgaaggcga ga 22
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cagattctcc tccgaaatgc cg 22
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaagatcttc gcacgcccaa ctcgacccct 30
<210> 11
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aactgcagaa ccaatgcatt ggcgccgcca actacgaaaa ca 42