CN112831506B - Yellow phyllotreta striolata cytochrome P450 gene and application thereof - Google Patents

Abstract

The invention discloses a phyllotreta striolata cytochrome P450 gene and application thereof, wherein the nucleotide sequence of the phyllotreta striolata cytochrome P450 gene is shown as SEQ ID NO:1, wherein the similarity between the gene sequence and other known P450 homologous genes is lower than 80%; the invention selects a 250bp gene fragment as a target point, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO:2, the similarity between the fragment sequence and other homologous genes in NCBI database is lower than 73%. Cloning a phyllotreta striolata cytochrome P450 gene fragment, connecting the fragment with an L4440 vector, transferring the recombinant plasmid into an escherichia coli HT115 competent cell, and obtaining dsRNA corresponding to a target fragment through IPTG induction, wherein the phyllotreta striolata can be effectively killed by feeding food containing the dsRNA, the phyllotreta striolata mortality rate can reach 61.5%, and the method can be applied to the prevention and treatment of Huang Qutiao phyllotreta striolata.

Description

Yellow phyllotreta striolata cytochrome P450 gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a phyllotreta striolata cytochrome P450 gene and application thereof.

Background

Yellow flea beetles Phyllotreta striolata (Fabricius) are Coleoptera (Coleoptera), phyllotoxin pests, which are mainly harmful to cruciferous vegetables, such as cabbage, cabbage mustard, rape, cabbage, etc. The life of the plant is four stages, namely four stages of eggs, larvae, adults and pupae, wherein the larvae live in soil and mainly eat root and bark of the plant, so that the phenomena of seedling missing and ridge breaking are often caused, and the larvae mainly eat tender leaves of cruciferous plants.

At present, research on phyllotreta striolata prevention and treatment at home and abroad mainly focuses on chemical prevention and treatment, physical prevention and treatment, biological prevention and treatment and transgenic technology. The method has the advantages that the plant secondary substances of the non-feeding hosts are used as one direction for preventing and controlling the phyllotreta striolata, and the mixed reagent of other plant secondary substances and chemical pesticides is extracted to have a certain effect on preventing and controlling the phyllotreta striolata. In crop cultivation, rotation of cruciferous plants with non-cruciferous plants is also a common method in fields for controlling phyllotreta striolata, but the control effect of these methods is not ideal. Spraying pesticide to cruciferous vegetables can only aim at pests on the ground, but cannot act on larvae in soil, so that the larvae, pupae and eggs in soil cannot be killed, the symptoms and root causes cannot be treated, serious environmental pollution can be caused, the quality of vegetables is reduced, and the physical health of people is affected. At the same time, abuse of the insecticide may lead to resistance of phyllotreta striolata. Therefore, a new technology for preventing and controlling the phyllotreta striolata effectively without causing harm to the environment is important. The transgenic insect-resistant technology improves the insect-resistant character of crops by expressing the insect-resistant toxin gene in plants, has the advantages of broad spectrum, high resistance and the like, but also induces the insect to generate resistance, has low specificity, has larger potential safety hazard to environment and biological groups, and is not accepted by consumers consistently.

Along with the development of molecular biotechnology, RNAi technology is increasingly applied to the field of insect control, and a good control effect is obtained in tests of insects such as diptera, hymenoptera and the like at present. In diptera mosquitoes, the gene Defensin of anopheles gambiae antibacterial peptide is silenced by using RNAi technology, and the function of the gene is identified, so that a new prey for researching the gene function of mosquitoes by using RNAi technology is drawn. The most studied hymenoptera insects using RNAi technology are italian bees, and the expression of the adult genes of the italian bees was successfully reduced by constructing dsRNA of the relevant genes.

Cytochrome P450 is widely distributed, and reported researches on P450 genes are mainly focused on economic insects, sanitary insects and agricultural insects such as houseflies, silkworms, cotton bollworms, red-mimetic theft, drosophila melanogaster, aedes aegypti, paecilomyces melanogaster, aphids and the like. However, the study of the phyllotreta striolata cytochrome P450 gene and RNAi thereof has not been reported. The invention obtains a cytochrome P450 gene highly expressed in the flea beetle by transcriptome sequencing of the phyllotreta striolata, and discovers that the similarity of the gene sequence and other known P450 homologous genes is lower than 80% by BLAST analysis on NCBI database. We selected a 250bp gene fragment as the target for constructing RNAi expression vectors. Through sequence alignment analysis, the similarity between the fragment sequence and other homologous genes in NCBI database is found to be lower than 73%, which indicates that the target has very high specificity. The expression condition of dsRNA is further optimized by cloning the phyllotreta striolata cytochrome P450 gene fragment and constructing an RNAi expression vector, and feeding proves that the phyllotreta striolata can be effectively killed, the death rate of the phyllotreta striolata can reach 61.5%, and the phyllotreta striolata death rate is obviously higher than that of a control group, so that the phyllotreta striolata can be applied to the control of Huang Qutiao phyllotreta striolata.

Disclosure of Invention

In order to solve the problems set forth in the background art, the present invention aims to provide a phyllotreta striolata cytochrome P450 gene and an application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a phyllotreta striolata cytochrome P450 gene has a nucleotide sequence shown in SEQ ID NO: 1.

A phyllotreta striolata cytochrome P450 gene fragment has a nucleotide sequence shown in SEQ ID NO: 2.

A method for obtaining a phyllotreta striolata cytochrome P450 gene fragment comprises the following steps of: 1, by using the plasmid as a template and an upstream primer SEQ ID NO:3 and a downstream primer with a cleavage site Sac I SEQ ID NO:4, carrying out PCR amplification to obtain the phyllotreta striolata cytochrome P450 gene fragment.

A dsRNA expression vector containing a phyllotreta striolata cytochrome P450 gene fragment comprises the following construction method: double enzyme digestion is carried out on vector plasmid capable of producing dsRNA and phyllotreta striolata cytochrome P450 gene fragment respectively under the conditions of Fast digest Kpn I and Fast digest Sac I and 37 ℃;

carrying out 1.2% agarose gel electrophoresis on a vector plasmid enzyme cutting product capable of generating dsRNA, then cutting gel to recover large fragments, and removing small fragments;

purifying the enzyme digestion product of P450 by using a common DNA product purification kit;

ligating the vector plasmid product recovered by cutting gel at 25 ℃ and capable of producing dsRNA with the P450 product obtained by purification;

preferably, the vector plasmid from which dsRNA can be produced is the L4440 plasmid.

The expression vector is constructed to obtain a high-efficiency expression system.

The preparation method of the high-efficiency expression system comprises the step of transfecting the expression vector into a host cell.

Further, the host cell is an RNaseIII-deleted host cell, preferably the RNaseIII-deleted host cell is an HT115 (DE 3) competent cell.

A synthesis method of dsRNA of a phyllotreta striolata cytochrome P450 gene fragment comprises the step of inducing the expression system or the prepared expression system to obtain the dsRNA corresponding to a target fragment through IPTG.

dsRNA of the phyllotreta striolata cytochrome P450 gene fragment synthesized by the method.

The application of the dsRNA of the phyllotreta striolata cytochrome P450 gene fragment in killing phyllotreta striolata is disclosed.

The beneficial effects of the invention are as follows: the nucleotide sequence of the phyllotreta striolata cytochrome P450 gene is shown in SEQ ID NO:1, wherein the similarity between the gene sequence and other known P450 homologous genes is lower than 80%; the invention selects a 250bp gene fragment as a target point, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO:2, the similarity between the fragment sequence and other homologous genes in NCBI database is lower than 73%. Cloning a phyllotreta striolata cytochrome P450 gene fragment, connecting the fragment with an L4440 vector, transferring the recombinant plasmid into an escherichia coli HT115 competent cell, and obtaining dsRNA corresponding to a target fragment through IPTG induction, wherein the phyllotreta striolata can be effectively killed by feeding food containing the dsRNA, the phyllotreta striolata mortality rate can reach 61.5%, and the method can be applied to the prevention and treatment of Huang Qutiao phyllotreta striolata.

The stability of dsRNA at room temperature is studied, and the result shows that the dsRNA is not degraded even when the dsRNA is placed for 72 hours at room temperature, so that the problem of how to obtain dsRNA which is rich in content and easy to produce and use is solved, and the input is low, and the dsRNA can be efficiently prepared in a large quantity by a high-pressure cell disruption method.

Drawings

FIG. 1 shows the results of P450 amplification, where M: DL5000 DNA marker;1: cytochrome P450 gene fragments;

FIG. 2 is a graph of the L4440 plasmid together with the multiple cloning site and cleavage results, wherein FIG. 2A is a graph of the L4440 plasmid together with the multiple cloning site; FIG. 2B shows the results of cleavage of the L4440 plasmid by SacI and KpnI, wherein M: DL5000 DNA marker; 1.2, 3: l4440 plasmid;

FIG. 3 is a colony PCR identification, wherein M: DL2000 DNA marker; 1.2, 3, 4, 5, 6, 7: recombinant plasmid L4440-P450;8: a negative control;

FIG. 4 shows the results of dsRNA and DNase I and RNase A digestion assays, wherein M: DL2000 DNA markers; 1: subjecting the extract to DNase I digestion; 2: subjecting the extract to RNase A digestion; 3: subjecting the extract to DNase I and RNase A digestion; 4: untreated extract control group;

FIG. 5 is the effect of different conditions of IPTG on induction of dsRNA expression, where M: DL2000 DNA markers; 1.2, 3: the induction time of 0mM IPTG is 2h,3h and 4h respectively; 4. 5, 6: induction time of 0.2mM IPTG was 2h,3h,4h, respectively; 7. 8, 9: induction time of 0.4mM IPTG was 2h,3h,4h, respectively; 10. 11, 12: induction time of 0.8mM IPTG was 2h,3h,4h, respectively;

FIG. 6 is a stability assay for dsRNA, wherein M: DL2000 DNA markers; 1-6: the dsRNA is placed for 0h, 12h, 24h, 36h, 48h and 72h at room temperature respectively; arrows indicate dsRNA band positions;

figure 7 is a dsRNA insecticidal mortality statistic.

Detailed Description

For a better understanding of the present invention, the following description will further explain the present invention in conjunction with specific embodiments, but the present invention is not limited to the following examples.

Experimental materials

The yellow flea beetle adults are obtained from Shenzhen city Long Huaji, mainly in the heart and leaf surface positions of cruciferous vegetables. It was placed in a culture chamber (temperature maintained at 28 ℃, relative humidity 75%, illumination intensity set at 60% lx, period L: d=14:10) and fed with fresh young leaves of chinese cabbage.

The main reagents required in the experiments include Trizol; DNA Marker DL2000; DNA Marker DL5000;11 XDNA/RNA Loading buffer; t4 DNA Ligase; 10×t4 DNA Ligase buffer; 10X Fast Digase green buffer; sacI (Fast Digase); kpnI (FastDigase); isopropyl alcohol; 75% ethanol; chloroform; DEPC water; deionized water (ddH 2O); liquid nitrogen; the easy script One-Step gDNA Removal and cDNA Synthesis SuperMix kit; a general DNA product purification kit; HT115 (DE 3) competent cells.

Since the wild type E.coli carries RNase III in its own gene, this enzyme recognizes and cleaves dsRNA, the method of producing dsRNA by induction expression of wild type E.coli is not feasible. In contrast, HT115 (DE 3) is a defective E.coli, does not have RNase III and thus can be used to express dsRNA, and furthermore, L4440 contains the bi-directional T7 promoter and the lac lactose operon, so that the recombinant plasmid L4440-P450 was transformed by HT115 competent cells and thus was studied herein to express dsRNA.

EXAMPLE 1 extraction of total RNA from phyllotreta striolata and Synthesis of cDNA

About 30 yellow flea beetles are taken, washed clean by ddH2O, sucked dry by filter paper, quickly ground by adding liquid nitrogen into the yellow flea beetles until the yellow flea beetles are white powder, and the yellow flea beetle total RNA is extracted by a Trizol method. The yellow flea beetle total RNA is subjected to reverse transcription by using an easy script One-Step gDNA Removal and cDNA Synthesis SuperMix kit to obtain single-stranded cDNA, and the nucleotide sequence of the single-stranded cDNA is shown as SEQ ID NO: 1. The expression level of the phyllotreta striolata cytochrome P450 gene (c 13467 _g1_i1) is high, the similarity of the gene and other known P450 homologous genes is lower than 80%, and the gene has strong specificity and is a phyllotreta striolata specific gene. The high-efficiency expression of the gene is very important for the metabolism and growth and development of the flea beetles, and the RNAi silencing of the gene reduces the expression quantity of the gene, and influences the normal metabolism and growth and development of the flea beetles, so that the effect of killing the pests can be achieved. And the gene has strong specificity, so that the gene generally cannot cause harm to other insects or other organisms.

EXAMPLE 2 cloning of the yellow flea beetle cytochrome P450 Gene

Primers were designed using Primer5.0 software, and KpnI and SacI cleavage sites were added to the 5 'end of the upstream primer and the 5' end of the downstream primer, respectively (P450 primer with cleavage site, GGTACC is KpnI cleavage site, GGTACC is SacI cleavage site are shown in the following Table). The cDNA obtained by reverse transcription is used as a template, and a primer PCR amplification with enzyme cutting sites (Kpn I, sac I) is used to obtain a yellow flea beetle cytochrome P450 gene fragment, the size of which is about 250bp, and the similarity of the gene fragment and other known homologous genes is lower than 73 percent as shown in figure 1. The PCR reaction system is as follows: 25 [ mu ] L2X TransTaqHiFi PCR SuperMix II, 1 ul each for the upstream and downstream primers, 2 ul for the template, and 50 ul for ddH 2O: pre-denaturation at 94℃for 5 min, denaturation at 94℃for 30 s, annealing at 55℃for 30 s, extension at 72℃for 1min,36 cycles, and extension at 72℃for 5 min.

P450-F	GGGGTACCACTCGAAGGATCTTGAGTACAT	SEQ ID NO：3
			P450-R	CGGTACCATGACGGCAACTGTAGGTCCCGA	SEQ ID NO：4

EXAMPLE 3 construction of phyllotreta striolata P450 interference vector

The L4440 plasmid and the P450 PCR recovered products were double digested for 2h at 37℃using Fast digest Kpn I and Fast digest Sac I, respectively. Carrying out 1.2% agarose gel electrophoresis on the L4440 plasmid enzyme digestion product, then cutting gel to recover large fragments, and removing small fragments; the cleavage products of P450 were purified using a common DNA product purification kit. The L4440 product recovered by ligation and the P450 product obtained by purification were reacted at 25℃for 2 hours, and HT115 (DE 3) competent cells were transformed from the ligation product, cultured for 60 minutes at 37℃on a thermostatic incubator shaker at 250 r/min, then plated on LB solid medium (100 mg/L AMP), and cultured overnight in a thermostatic incubator at 37℃until colonies grew. White colonies are selected, colony PCR identification is carried out on the white single colonies, positive monoclonal sequencing which is successfully screened and identified is carried out, sequencing results are compared, and L4440-P450 recombinant plasmids which are successfully constructed are selected.

The L4440 plasmid was 2790bp in size and itself harbored a bi-directional T7 promoter as shown in FIG. 2A. In this experiment, sacI and KpnI were selected for double cleavage at multiple cloning sites. Agarose gel electrophoresis analysis shows that the L4440 plasmid is subjected to double enzyme digestion to generate two fragments, the size of a large fragment is about 2700bp, the size of a small fragment is about 138bp, the brightness of a small fragment gene on an electrophoresis chart is very low, the efficiency of enzyme digestion products is relatively good, the enzyme digestion products are subjected to gel digestion recovery, the large fragment products are reserved, and the small fragment products are removed, as shown in figure 2B.

The P450 PCR amplification product was subjected to double cleavage and the product was purified. Then, the two products were ligated under a ligation system, followed by transformation of the ligation products into E.coli HT115 competent cells, and resistance selection and sequencing alignment were performed, as shown in FIG. 3, and the transformed ligation plasmid was identified by colony PCR to conform to the expected results, indicating successful construction of the recombinant plasmid, designated as L4440-P450 plasmid.

EXAMPLE 4 dsRNA-induced expression and purification

The L4440-P450 recombinant plasmid was transformed into HT115. Taking 20 mu L to 10mL of fresh LB liquid culture medium (containing 100mg/L AMP), shake culturing for 16h in a constant temperature culture shaking table at 37 ℃ and 220r/min, sucking 2mL of cultured bacterial liquid, adding into a conical flask containing 100mL of fresh liquid culture medium (containing 100mg/L AMP), culturing to logarithmic growth phase, wherein at the moment, OD600 = 0.5 or so, and shake culturing time is generally 3 to 4h. Adding IPTG for induction, and placing the mixture into a constant temperature culture shaking table with the final concentration of 0.4mM, and continuously carrying out shake culture at 37 ℃ and 220r/min for 4 hours. dsRNA was extracted by Trizol method and 30 μl ddH2O was added. An appropriate amount of the above samples were aspirated, and digested with DNase I and RNaseA, respectively, to identify dsRNA, and the agarose gel electrophoresis results are shown in FIG. 4. DNase I is mainly used to degrade DNA molecules in the extract, whereas RNase A digests single stranded RNA. The dsRNA extracted by Trizol is still stable after being treated by DNase I and RNase A in sequence, which shows that the dsRNA is successfully obtained.

Example 5 optimization of dsRNA Induction conditions

dsRNA is induced by IPTG, so that gradient experiments can be carried out from the aspects of the concentration of the IPTG and the culture time to select induction conditions suitable for high-efficiency expression of the dsRNA. Firstly, the concentration of IPTG is adopted, HT115 bacterial liquid transformed by L4440-P450 recombinant vector is sucked to be 1:50 were added to 4 conical flasks, each containing 100mL of fresh LB liquid medium (100 mg/L AMP), and the final concentrations of IPTG were set at 0mM, 0.2mM, 0.4mM, 0.8mM, respectively. Then, when od600=0.5, IPTG was added, and the induction culture time of IPTG was set to 2h,3h, and 4h, respectively. IPTG at different concentrations was induced to culture for three different times, respectively. Other conditions were unchanged.

dsRNA extracted at different concentrations and at different times is firstly stored in a refrigerator at the temperature of minus 20 ℃, and after all the dsRNA is extracted, the expression quantity of the dsRNA induced at different times by different IPTG concentrations is detected through agarose gel electrophoresis.

As can be seen from fig. 5, the bacterial liquid without IPTG induction cannot produce dsRNA; when IPTG concentration is low (0.2-0.4 mM), different induction times under the same conditions of IPTG concentration lead to bands of different brightness, and as time increases, the brightness of the bands increases, which indicates that the expression amount of dsRNA increases with time, wherein at 4h, the expression amount of dsRNA is the largest (6 and 9 in fig. 5), but when IPTG concentration is high (0.8 mM), the expression amount of dsRNA is the largest at 3h of induction (11 in fig. 5); when the IPTG induction time is the same, the expression quantity of the IPTG induction dsRNA with different concentrations is different, and the expression quantity of the dsRNA is relatively higher under the induction of 0.2-0.4mM IPTG. Thus, induction for 4h with 0.2-0.4mM IPTG was considered to produce a relatively high expression level of dsRNA, depending on the results of optimization of the dsRNA induction conditions.

Example 6 dsRNA stability assay

Crushing bacterial liquid induced by IPTG by using a high-pressure cell crusher, carrying out crude extraction on dsRNA by isopropanol precipitation, then placing at room temperature for 0h, 12h, 24h, 36h, 48h and 72h, absorbing a part of dsRNA in each time period, preserving at-20 ℃, and finally carrying out agarose electrophoresis detection together, and comparing and judging the stability of the dsRNA at room temperature.

From fig. 6, it can be found that the dsRNA is not degraded at room temperature, which indicates that the dsRNA can remain stable at room temperature, so that subsequent studies on anti-yellow stripe flea beetle experiments can detect the insecticidal effect by spraying the crushed bacterial liquid.

EXAMPLE 7 dsRNA insecticidal assay

dsRNA feeding is mainly carried out by feeding artificial feed to insects, crops expressing dsRNA, strains having an effect of inducing dsRNA, or spraying dsRNA directly on the surface of crops. The feeding method is simple to operate, low in input cost and easy to realize, and the insect pest can enter the insect pest body in the most original natural input mode, so that the purpose of silencing genes is achieved.

Diluting with sterile water to prepare P450 dsRNA solution with concentration of 200ng/ml, soaking tender leaf of vegetable heart in P450 dsRNA solution and control solution (ddH 2O) for one to two minutes respectively to make the surface of tender leaf fully contacted with dsRNA solution, and updating the soaked leaf every 24 hours for feeding phyllotreta striolata, observing and recording the growth state and the statistical death rate of phyllotreta striolata. Three replicates were set up for each of the control and test groups, with 20 phyllotreta striolata adults in each replicate used for the feeding experiment.

The tender leaves of the cabbage heart soaked by the ddH2O and the P450-dsRNA are observed, and the yellow leaf-flea beetle in the control group and the test group is not obviously affected in growth and development at the first three to four days, the test group starts to show RNAi effect on the fifth day, the yellow leaf-flea beetle dies appear in succession, the P450 dsRNA shows obvious inhibition effect on about 15 days, the death rate of the test constituent insects reaches more than fifty percent, the average death rate reaches 61.5 percent, and the death rate is obviously higher than that of the control group, as shown in figure 7.

The RNAi process is completed in several steps. The first step is the initiation phase of RNAi, the exogenously introduced dsRNA is cleaved by endonuclease Dicer into 21-25 bp fragment siRNA; the second step is the assembly phase of siRNA, the siRNA is loaded to RISC complex under the action of Argonaute enzyme, etc., after the complex is formed, one strand of double-stranded RNA is degraded, and the single-stranded RNA loaded and activated on RISC complex is called guide strand; the third step is the effector phase of RNAi, where the siRNA entering the RISC complex finds and binds to the target mRNA sequence in a sequence complementary manner, resulting in translational inhibition of the target mRNA or cleavage thereof into short fragments of RNA by the action of some endonuclease, thereby causing a silencing effect on the target gene.

RNAi control of pests is mainly the application of RNAi in non-cellular autonomy. Non-cellular autonomous RNAi includes environmental RNAi, which is the uptake of dsRNA by cells from the external environment, and systemic RNAi, which is the transport of silencing signals within multicellular organisms between cells or tissues. Spraying dsRNA of a target gene on crop leaves, wherein pests take the leaves to enable the dsRNA to reach midgut of the pests and be absorbed by the pests, and the process is environment RNAi; and after dsRNA is absorbed by the midgut cells, the silencing signal reaches the expression cells or tissues of the target gene through the systemic RNAi transport process, and the specific silencing of the target gene is induced.

The foregoing is merely a specific embodiment of the present invention and not all embodiments, and any equivalent modifications of the technical solution of the present invention that will be obvious to those skilled in the art from reading the present specification are intended to be encompassed by the claims of the present invention.

SEQUENCE LISTING

<110> Shenzhen university

<120> A yellow flea beetle cytochrome P450 gene and application thereof

<130> CP119011134C

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 1893

<212> DNA

<213> artificial sequence

<400> 1

cacgtataaa tagtataaaa agaaaggaga tcttcacacc gtcgttagtt tgaggttgcg 60

tctcaaaaag caaaatgacg gcaactgtag gtcccgacca aatcagtcaa aatgcagtcg 120

tatctgccag cagtgttttt tatttcctgc tgctgccggc ccttgcccta ttctacgcct 180

actggaagat ttcccgtcgc catctgctcg aattggcaag caaaatcccc ggtcctgaag 240

gttaccccat cataggaaat gcattggact ttgttggaag ctcgcacgac attttcaaaa 300

aaatgtactc aagatccttc gagttcggca aaatcgccaa attttgggcc ggaccgaaac 360

tcttgatttt cttaatcgac ccgagagacg tggaaatcat tctcagcagt cacgtgcaca 420

tcgataaggc cagcgaatac aaattcttca aaccgtggtt gggcgacggt cttctcatct 480

ccaccggcca gaaatggagg gctcacagga aactcattgc gcccactttc catttgaacg 540

tgctcaaatc cttcatcgac ctgttcaacg ccaactcccg ggaggtcgtt caaaagttga 600

aaaaggaagc tggaaaggaa ttcgactgtc acgactacat gtccgaagct accgttgaaa 660

ttttattgga aactgctatg ggagtgagca agaaaactca agacaagagc ggatacgact 720

acgccttagc tgtaatgaaa atgtgcgata ttttgcacat tcgtcacacc aagatttggc 780

tcagacctga catcctattc aatttaacaa aacacgccac gaaccaaaaa ggattgctca 840

acaccattca cagcttgacc aggaaggtga tcaagaagaa gagagcagat ttcgaaaaag 900

gaatccgtgg atcaactgct gaagtacccg aagatgctaa aacccaaaaa tacgaaaaga 960

acgtcagttc taaaaccgtc gtcgaaggtc tttcctacgg acaatccgcc ggtcttaagg 1020

atgatttgga tgtcgatgat gatgtaggtg aaaagaagag aatggccttc ttggacttgt 1080

taatcgaagc atcccaaaac ggcgttgtca ttaacgatga agaaattaag gaacaagtag 1140

acaccattat gttcgaaggt cacgatacaa ctgctgccgg tagcagtttc ttcctttcga 1200

tgatgggagt tcaccaagac atccaagaca gagttatcca agaaattgac gaaatctttg 1260

gcgactctga caggccagcc accttcgccg atactttaga aatgaaatac ttggaaagat 1320

gcttaatgga aacccttcgt ttgtacccac cagtaccgat tatcgctagg caactcaagc 1380

aggatgtaaa attagcttct ggtgactaca ctttaccatc tggtgccact atcatcattg 1440

gaaccttcaa gattcaccgt gatgctgaca cttaccccaa ccctgagaag tttgaccctg 1500

acaatttctt gccagagcgt actgcaaaca ggcactacta ctccttcatt ccgttctcag 1560

ctggacccag gagttgtgtc ggtcgtaaat acgctatgtt gaaactgaaa attctgcttt 1620

caactatctt gagaaactac agagttaaat ctaatctagg cgaggaagac ttcaaacttc 1680

aagctgatat tatcttgaag agggctgaag gattcaaaat taaattggaa cccaggaaac 1740

ctttgcttaa agcataaaaa tccagtgatc ctgattctac taatttataa taggaattaa 1800

gtgtttttga gtgttttgta atatatttag ttaattgtaa tccacagcaa tgatgaacaa 1860

gattcacaac taaaaaaaaa aaaaaaaaaa ata 1893

<210> 2

<211> 250

<212> DNA

<213> artificial sequence

<400> 2

actcgaagga tcttgagtac atttttttga aaatgtcgtg cgagcttcca acaaagtcca 60

atgcatttcc tatgatgggg taaccttcag gaccggggat tttgcttgcc aattcgagca 120

gatggcgacg ggaaatcttc cagtaggcgt agaatagggc aagggccggc agcagcagga 180

aataaaaaac actgctggca gatacgactg cattttgact gatttggtcg ggacctacag 240

ttgccgtcat 250

<210> 3

<211> 30

<212> DNA

<213> artificial sequence

<400> 3

ggggtaccac tcgaaggatc ttgagtacat 30

<210> 4

<211> 30

<212> DNA

<213> artificial sequence

<400> 4

cggtaccatg acggcaactg taggtcccga 30

Claims (3)

1. The application of the dsRNA of the phyllotreta striolata cytochrome P450 gene fragment in killing the phyllotreta striolata is characterized in that,

the synthesis method of the dsRNA of the Huang Qutiao flea beetle cytochrome P450 gene fragment comprises the following steps: constructing a dsRNA expression vector containing a phyllotreta striolata cytochrome P450 gene fragment to obtain a high-efficiency expression system, and inducing the expression system by IPTG to obtain dsRNA corresponding to the target fragment;

the nucleotide sequence of the Huang Qutiao flea beetle cytochrome P450 gene fragment is shown in SEQ ID NO:2 is shown in the figure;

the nucleotide sequence of the Huang Qutiao flea beetle cytochrome P450 gene is shown in SEQ ID NO:1 is shown in the specification;

the method for obtaining the Huang Qutiao flea beetle cytochrome P450 gene fragment comprises the following steps: to contain a nucleotide sequence as shown in SEQ ID NO:1, by means of an upstream primer with an enzyme cleavage site Kpn I as a template, the sequence of SEQ ID NO:3 and a downstream primer with a cleavage site Sac I SEQ ID NO:4, carrying out PCR amplification to obtain a phyllotreta striolata cytochrome P450 gene fragment;

the construction method of the dsRNA expression vector containing the phyllotreta striolata cytochrome P450 gene fragment comprises the following steps: double enzyme digestion is carried out on vector plasmid capable of producing dsRNA and phyllotreta striolata cytochrome P450 gene fragment respectively under the conditions of Fast digest Kpn I and Fast digest Sac I and 37 ℃;

carrying out 1.2% agarose gel electrophoresis on a vector plasmid enzyme cutting product capable of generating dsRNA, then cutting gel to recover large fragments, and removing small fragments;

purifying the enzyme digestion product of P450 by using a common DNA product purification kit;

ligating the vector plasmid product recovered by cutting gel at 25 ℃ and capable of producing dsRNA with the P450 product obtained by purification;

the vector plasmid capable of producing the dsRNA is L4440 plasmid;

the preparation method of the high-efficiency expression system comprises the step of transfecting the expression vector into a host cell.

2. The use of claim 1, wherein the host cell is an RNaseIII-deleted host cell.

3. The use of claim 2, wherein the RNaseIII-deleted host cell is an HT115 (DE 3) competent cell.