CN108823212B - American cockroach amputation regenerates correlation Wingless signal path gene and its application of dsRNA - Google Patents

Abstract

The invention discloses the applications of American cockroach amputation regeneration correlation Wingless signal path gene and its dsRNA, American cockroach amputation regeneration associated genes are respectively Wg, Arr, Dsh, wherein, the nucleotide sequence of Wg is as shown in SEQ ID No.1, the nucleotide sequence of Arr is as shown in SEQ ID No.2, and the nucleotide sequence of Dsh is as shown in SEQ ID No.3.By designing the dsRNA of targeted silent American cockroach amputation regeneration associated genes, the prevention and treatment to American cockroach may be implemented.

Description

Expression of Wingless signaling pathway genes and their dsRNA related to limb regeneration in Periplaneta americana application

technical field

The invention belongs to the field of genetic engineering, and in particular relates to the application of genes related to the Wingless signaling pathway in the regeneration of amputated limbs of Periplaneta americana and dsRNA thereof.

Background technique

Cockroaches are native to Africa and live in a humid, warm and dark place; they like to hide in hidden crevices; their activity time is concentrated at night, so it is difficult for humans to eliminate their populations. The American cockroach is a relatively common cockroach. It is an omnivorous insect. It can eat sweet and sour foods, insect corpses, etc. It prefers sweets. It can survive for many days without water and food; at the same time, the American cockroach is very destructive, damaging clothing and spreading diseases. It is a common household hygiene pest. Periplaneta americana larvae have a strong ability to regenerate appendages (legs and antennae, etc.), and their survival ability is extremely strong, so it is extremely difficult to control.

Since the 1940s, chemical pesticides and organic pesticides have shown efficient insecticidal effects in pest control practices, bringing revolutionary changes to pest control. However, with its long-term and extensive use, a series of negative problems such as pesticide residues, water and environmental pollution, disruption of biological chains, and ecological imbalances have become increasingly prominent. At present, the products used for cockroach control in the market are mainly chemical pesticides. With the strengthening of people's attention to the sanitary environment and the emergence of the problem of cockroach resistance, the development of environmentally friendly, efficient and non-toxic cockroach control methods has become critical to national economic development. , key issues of social progress and food safety. The use of advanced biological control techniques (RNA interference (RNAi)) for pest control is increasing.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is: how to establish a new, safe and efficient method for preventing and controlling Periplaneta americana.

The technical scheme of the present invention is as follows: the genes related to the amputation and regeneration of Periplaneta americana are Wg, Arr and Dsh, wherein the nucleotide sequence of Wg is shown in SEQ ID No. 1, and the nucleotide sequence of Arr is shown in SEQ ID No. 1. As shown in SEQ ID No.2, the nucleotide sequence of Dsh is shown in SEQ ID No.3.

A group of dsRNAs targeting the silencing of the amputated limb regeneration-related genes of Periplaneta americana as claimed in claim 1 , the dsRNAs are three, respectively targeting and silencing three genes of Wg, Arr and Dsh, wherein the dsRNA targeting and silencing the Wg gene The nucleotide sequence of dsRNA is shown in SEQ ID No.13, the nucleotide sequence of dsRNA targeting silencing Arr gene is shown in SEQ ID No.14, and the nucleotide sequence of dsRNA targeting silencing Dsh gene is shown in SEQ ID No.15 shown.

The above-mentioned preparation method of a group of dsRNA targeting and silencing the amputated limb regeneration-related gene of Periplaneta americana described in claim 1, the dsRNA targeting and silencing Wg gene is prepared by the following method: The gene fragment was cloned into the pTOPO vector, named pTOPO-Wg, then pTOPO-Wg was used as the template, and the primers SEQ ID No.7 and SEQ ID No.8 containing the T7 promoter at both ends were used for PCR amplification, and the PCR product passed through T7RiboMAX ^TM Express RNAi System was transcribed and synthesized; the dsRNA targeting silencing Arr gene was prepared by the following method: the gene fragment shown in SEQ ID No. 5 was cloned into pTOPO vector, named pTOPO-Arr, and then pTOPO-Arr was used as a template, Carry out PCR amplification with primers SEQ ID No. 9 and SEQ ID No. 10 containing T7 promoters at both ends, and the PCR product is transcribed and synthesized by T7RiboMAX ^™ Express RNAiSystem; the dsRNA targeting silencing Dsh gene is prepared by the following method: The gene fragment shown in ID No. 6 was cloned into the pTOPO vector, named pTOPO-Dsh, and then using pTOPO-Dsh as a template and primers SEQ ID No. 11 and SEQ ID No. 12 containing T7 promoter at both ends, the PCR amplification, the PCR product was transcribed and synthesized by T7RiboMAX ^™ ExpressRNAi System.

The nucleotide sequences of the amputated limb regeneration-related genes of Periplaneta americana targeted by dsRNA are shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively.

A recombinant vector or recombinant bacteria containing the nucleotide sequence shown in SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6.

Application of the above-mentioned group of dsRNAs targeting the silencing of the above-mentioned genes related to the regeneration of amputated limbs of Periplaneta americana in the prevention and treatment of Periplaneta americana. The above-mentioned dsRNA was injected into the body cavity of American cockroach, and the results showed that the synthesized dsRNA could specifically silence the mRNA expression of Wg, Arr and Dsh genes of American cockroach, and lead to the aplastic disorder of amputated limbs in American cockroach.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention designs a pest control scheme aiming at the current situation that Periplaneta americana has tenacious regeneration ability of amputated limbs and is difficult to prevent and control, which not only can effectively eliminate pests, but also avoids environmental pollution, and is safe and reliable. An important process of regeneration of amputated limbs in cockroaches is the shaping of the regenerated legs, and the wingless signaling pathway includes several important morphogen genes, such as Wg, Arr and Dsh, etc. The absence of this pathway will prevent cockroaches from normal appendage regeneration. , using its important biological functions for pest control, saving time and effort, safe and efficient.

Description of drawings

Figure 1: Flow chart of sample processing for transcriptome sequencing of Periplaneta americana;

Figure 2: The expression heat map of Wingless signaling pathway genes related to the regeneration of amputated limbs of Periplaneta americana;

Figure 3: Effects of Wg, Arr and Dsh gene dsRNA treatment on the regeneration of amputated limbs of Periplaneta americana;

Figure 4: Detection of Wg, Arr and Dsh gene silencing efficiency.

Detailed ways

Example 1 Screening of Wingless signaling pathway genes related to amputated limb regeneration

1) Preparation of RNA-Seq samples

The American cockroach was selected, amputated, and leg tissues were collected. The specific amputation treatment method was as follows: Amputation was performed on the joint of the hind limb trochanter and leg segment on one side, and the complete basal segment and trochanter were preserved. The leg, tibia and tarsus were cut off together (Fig. 1), and the other leg was all reserved for the control experiment. The amputation method was adopted based on the inventor's discovery that the trochanter played a very important role in the regeneration of the amputated limb. When the trochanter was cut off, new legs with normal shape and function could not be regenerated; samples were collected at different time points after amputation (1dpa, 3dpa, 5dpa, 7dpa, and 10dpa (day postamputation, dpa)) (Figure 1 ); then the trochanteric and basal ends of the amputated side were collected as the experimental group, and the tissue symmetrical to the experimental group was taken as the control group, named Pa.T 1/3/5/7/10dpa and Pa.CK 1/3 respectively /5/7/10dpa, each set of experiments contained three biological replicates. Total RNA was extracted from the collected tissues and handed over to Beijing Biomaike Biotechnology Co., Ltd. for second-generation transcriptome sequencing (RNA-Seq). The platform selected was IlluminaHiSeq. After the completion of Unigene functional annotation (NR, Swissprot, GO, EggNOG, Pfam, KEGG) and gene structure analysis (CDS prediction; SSR analysis), gene expression analysis was performed at the mRNA level by comparing with the untreated group (gene expression Quantitative and overall distribution analysis of gene expression in different samples), differentially expressed gene screening, differentially expressed gene cluster analysis, functional annotation and enrichment analysis of differentially expressed genes (differentially expressed genes GO function enrichment, differentially expressed genes EggNOG classification, differentially expressed genes Expression gene KEGG annotation, differentially expressed gene KEGG pathway enrichment analysis) and differentially expressed gene-protein interaction network, etc., and finally screened out several signaling pathways and genes related to tissue damage repair and amputated limb regeneration. Fixed expression mode for regeneration time nodes.

Based on the P. americana genome database, bioinformatics methods were used to search for Wg, Arr and Dsh genes in the Wingless signaling pathway of P. americana. After sequence analysis and alignment, the Wg, Arr genes in the Wingless signaling pathway of P. americana were obtained. and Dsh gene sequences, the cDNA nucleotide sequences are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively, the full lengths are 1194bp, 5052bp and 3180bp, respectively, encoding 398 amino acids, 1684 amino acids and 1060 amino acids.

Example 2 Synthesis and acquisition of dsRNA targeting Wingless signaling pathway genes

1) Design of dsRNA primers for Wg, Arr and Dsh genes

Based on the sequences of Wg, Arr and Dsh genes obtained in this study, the dsRNA design website E-RNAi (https://www.dkfz.de/signaling/e-rnai3/) was used to target genes Wg, Arr and Dsh. The entire open reading frame sequence is copied and pasted into the website, and the optimal dsRNA targeting sequence is obtained by screening the design parameters. The best amplification primer sequences generated by the website were selected, and the dsRNA primers targeting the Wg gene were Wg.T7F: TAATACGACTCACTATAGGAGACGGCGTTCATATACGC (SEQ ID No.7) and Wg.T7R: TAATACGACTCACTATAGGTCCGGGTTGTAGGGTTTCA (SEQ ID No.8); amplification targeting The dsRNA primers for the Arr gene are Arr.T7F: GGATCCTAATACGACTCACTATAGGAACGGCACTCATACTGGACC (SEQ ID No. 9) and Arr.T7R: GGATCCTAATACGACTCACTATAGG TTTGTTGCTGGTCTTTGTCG (SEQ ID No. 10); the dsRNA primers for amplifying the Dsh gene are Dsh.T7F: GGATCCTAATACGACTCACTATAGGACATGTCCAATGATGAGGCA (SEQ ID No. 11) and Dsh.T7R: GGATCCTAATACGACTCACTATAGG TGTGAAGCATATTTGCGAGC (SEQ ID No. 12). All primers contained the T7 promoter sequence (GGATCCTAATACGACTCACTATAGG), and were synthesized by Qingke Biological Co., Ltd.

2) Synthesis of double-stranded RNA (double strands RNA, dsRNA)

Total RNA from the legs of Periplaneta americana was extracted using the TRIzol (Life technologies) method, followed by reverse transcription using oligodT primers (PrimeScript II reverse transcriptase (Takara Bio, Shiga, Japan)) to synthesize the first cDNA strand. Using the primers designed above, a DNA fragment containing the target sequence was obtained by amplifying the cDNA as a template, and cloned into the pTOPO vector (Aidlab, China), and sequenced to verify whether there was a base mutation in the sequence, and selected the one without any mutation. The clones were used for subsequent studies, and the vectors were named pTOPO-Wg, pTOPO-Arr and pTOPO-Dsh, respectively.

Take the constructed pTOPO-Wg, pTOPO-Arr and pTOPO-Dsh vectors as templates respectively, and use the primers designed in step 1) to carry out PCR amplification to obtain a large number of PCR products containing T7 promoters at both ends, and their nucleotide sequences are as follows: SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 are shown. The RNA was recovered for in vitro transcription using a gel recovery kit (Omega). Then, T7RiboMAX Express RNAi System (Promega Corporation) was used to synthesize forward and reverse RNAs. After T7RNA polymerase and DNaseI were sequentially treated, the two forward and reverse RNAs were mixed and treated at 70°C for 10min, and then gradually cooled to room temperature to make it After annealing, it becomes a double strand to obtain dsRNA, the nucleotide sequences of which are shown in SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively. And the concentration was diluted to 3ug/ul.

Experimental Example 1 In vivo injection of dsRNA affects the regeneration of amputated limbs of Periplaneta americana

In order to verify the screened Wingless signaling pathway and the functions of Wg, Arr and Dsh genes in the regeneration of the amputated limbs of Periplaneta americana, the method of injecting dsRNA into the newly amputated Periplaneta americana was used. The gene expression interference function of dsRNA was used to verify the function of the gene, and the injection of CK, which does not target any gene of Periplaneta americana, was used as a negative control for dsRNA treatment. The general process is as follows: firstly select the newly molted cockroach larvae (d0), continue to cultivate for one day to adapt to the surrounding environment, and then give it the first dsRNA injection (d1), 24 hours later, one hind limb of the American cockroach was injected Amputation was performed (d2), the basal and trochanteric segments were preserved, the leg segment, tibia, and tarsus were clipped together, followed by three additional dsRNA interferences on the following d4, d7, and d14 days, each Inject 3ug. The specific details of the injection are as follows: Periplaneta americana larvae were anesthetized and fixed with tape, and then the dsRNA solution was injected into the abdomen of the American cockroach using the microinjection method. After injection, the effect of dsRNA treatment on the regeneration of amputated limbs in larvae was observed until the next molting. It was found that the decreased expression of Wg, Arr and Dsh genes significantly affected the regeneration ability of amputated fingers of American cockroach. The results are shown in Figure 3.

Experimental Example 2 Silencing detection of Wg, Arr and Dsh genes in Wingless signaling pathway

The silencing efficiency of Wg, Arr and Dsh was detected 24h after the second injection of dsRNA, and the leg tissues of Periplaneta americana were collected as RNA extraction objects, and each group was set up with 3 biological replicates. The total RNA of each sample was extracted and reverse transcribed into first-strand cDNA. Real-time PCR was used to detect the relative expression levels of target genes (Wg, Arr and Dsh) and housekeeping gene (β-actin), and the △△Ct method was used to calculate the target gene. Gene silencing efficiency. The results are shown in Figure 4, and it was found that dsRNA treatment could significantly reduce the expression of targeted genes and achieve the purpose of gene silencing.

The above-mentioned embodiments only represent specific implementations of the present application, and the descriptions thereof are specific and detailed, but should not be construed as limiting the protection scope of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application.

sequence listing

<110> Name of applicant: South China Normal University

<120> Application of Wingless signaling pathway genes and their dsRNA related to the regeneration of amputated limbs in Periplaneta americana

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gccgaggagg tggccagcaa gaacaaggcc gggcgcggac gcggcagcaa gtggtggggg 120

attgcgaaag ccggcgagcc caacaacttg cttccgcaga cgccgggcgc gctctacatg 180

gacccggccg tgcacgccat tctgcggcgg aagcagagac gtctagttcg ggagaacccg 240

ggagttcttg tggcggtagc caaaggtgct aaccaggcca tcgtagaatg ccagttccag 300

tttcgaaaca ggaggtggaa ctgctcgaca agaaattttc tacgaggcaa aaatctcttc 360

ggaaaaattg ttgacagagg ttgtcgggag acggcgttca tatacgcgat cacaagtgcg 420

ggcgtgacac acgccatcgc gcgggcgtgc agcgagggca gcatcgagtc gtgcacgtgt 480

gattacagcc accaggcgcg ggcgccgcag gtgacgtccg tgcccggcct gcgcgactgg 540

gagtggggcg gctgctccga caacatcggc tacggcttca agttctcccg cgaattcgtc 600

gataccggcg agcgggggcg caacctccgc gagaagatga atctccacaa caatgaggcc 660

ggcagagcgc acgtttcctc ggagatgcgt caagaatgta agtgtcacgg catgtctggc 720

tcctgcacgg tcaagacctg ctggatgcgg ctgcccagct tccgagtcgt aggcgacaac 780

ctcaaggacc gcttcgacgg cgcctccaga gtgatggtga gtaacgcggg cagcctgcgc 840

ggccagggtg gtagcggcgg cagcggcgtc ggtggtaaga agaacagata caacttccaa 900

ctgaaaccct acaacccgga ccacaagccg cccggcacca aagacctggt ctacttggag 960

ccttccccag ggttctgcga gcgcaacccg agactcggta tccaaggcac gcacggacgt 1020

cagtgcaacg atacgtcgat aggcgtggat ggttgcgacc tcatgtgttg tgggcgagga 1080

tatagaactc atgaggtgtc cgtggtgcag aggtgtgcgt gcatgttcca ctggtgctgc 1140

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<213> Periplaneta americana

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atgaagtgtg cctgcagccg aagaaacgag ttatgtcgca agagcaatcc aattattgta 60

gttaggatgt tagtggcgtc gtggatcgcc ctaataagct gtttgataac gtgcataaat 120

gcaaatccaa atctactttt ctcaacatca aaagatatta gagtcgtcag tgtgtcaaag 180

cccagcaagg tagtgacaat agttaaggac ttggaagaag gagcagctat agacttctat 240

tatgagggca acctggtgtg ttggactgat catgggcttg agatgatcca gtgtatttct 300

tataatggca catatattgg atctaaggtt gatgtagtta caacaggtct catctcacca 360

gctggtcttg cctgtgattg gttaacacaa aaattgtatt ggactgatgg cgagaccaat 420

cgtattgaag tggccaccct gaaaggagat catcggaaag ttcttttctg ggatgatatc 480

gatcaaccac gtgccatcac tgttgttcct atgaagagct taatgttttg gacagattgg 540

ggtgaaattc caaaaataga acgtgctggt atgaatggtg atcctactac tcgcaaagtt 600

attgtttctg aaaatatatt ttggccaaat ggtttgacag tggattatga cgcagagtta 660

gtatattggc tagatggaag actgaagttc gtggaagtca tggattacga gggtcgcaac 720

aggaggacta ttgtcaagga tggagttaac tacccatttg cattaaccat atttcagaat 780

aagctatact ggacagattg gaaaacatgg tcaatccacg tgtttgataa aacatctaat 840

gcacttccta aagagattat acatggtggt tatgttccaa tggacattca tgtttgggat 900

gctcaaagac aaccaccagg ggacacgcct tgccatcaca acaacggtgg atgctcacat 960

ctctgtcttc tagctcccta cccaccagga tacacgtgtg ctcaggagat gctcttgttg 1020

gtgcaaagaa ctgatatctg cacaatatca ttggattcac ctgattacac caactttgtg 1080

ctgccattgg ttggaattaa gcatgccatt gctatagatt acgaccctgt caatggatat 1140

ctttattgga ctgatgatga ggccagggca atacggagag ctcgtctaga tggatcagat 1200

caagaagacc ttatagtaac agaagttgaa catcctgatg gaattgctgt ggattgggtt 1260

gctcgaaatt tatattggac agacactgga actgatcgaa ttgaggtagc tagacttact 1320

ggagcttcga gaaaggtgct gattaacgaa gacttacatg aacctcgtgc cattgcactg 1380

gctccagaga agggatggat gttctggtcc gactggaatg acaaggttcc taagctagag 1440

cgtgcagctc ttgatggttc tgatcgtgta gtattgatta gcaaagatct tggttggcct 1500

aatggcattg ctttggatct tgataataat aaaatatatt ggtgtgatgc taagactgat 1560

aaaattgagg ttgccaacat ggctgatgga gaggatcgtc gtgaagttat tggtgacaac 1620

ttgccacatc ttttttggact gagtctcctt ggtgattacc tgtattggac agactggcaa 1680

agacgaagta ttgatcgagt aaaagtcagt ggggatggaa gagaagttat tgttgaccag 1740

atacccaatc tcatgggtct gaaggcaatt cgactgggtg aggttaaggg aaggaatccg 1800

tgcacagaaa ataatggaaa ctgcagccac ttatgcctca acaggccaaa caacaattac 1860

atctgcgcct gccaaattgg gtatgaattg gcagctaatg aaagaacgtg tgttgtacca 1920

gaggcattcc ttctctttgc acgaaaagag aatattggtc gcataagtat agaaaatgga 1980

aacaatgatg caattattcc tgtaaccgga gtgaaagatg ccagtgcttt ggattttgac 2040

atcaatgata atcgtatcta ctggacagac gtcaaagtga aagctataac acgtgccttt 2100

atgaatggat cagatatgga aaagatagtt gaatttggtt tggattctcc agaaggtatg 2160

gctgtggatt gggtggcgca taatatatat tggactgata cgggtagtaa gagaattgaa 2220

gtcgcccgct tgactggacg atcaaggaaa gttttgattt ggaaaggtat agatgaacct 2280

cgcagtttag ctctggatcc gagagaaggg ttcatgtatt ggagtgactg gggatccaag 2340

gggtccatat caaaagctgc aatggacgga actcaacgca gtatcattat agctaagtta 2400

ggacgtgcca atggcttgac gatagattat gttcagcatc gtctttattg gacagaactg 2460

gaacaacatg taatcgagag ctctgatcta gaagggaaac aaaggatgac aattattaat 2520

caaaatgttg aaaagccatt tggattaact cagtatttgg attatatcta ctgggctgac 2580

tggaaaactg gcaatattga gagggccaac aagacaaatg gcacaaacag gacagtgata 2640

cataggcatt tggaatcaat cacagatatc cttatttttc acaattcgcg acaatcagct 2700

tggaatcagt gtgctgtggg aaatggtgga tgctcgcacc tgtgtttggc attgccgtct 2760

gtctctaacc catctacagc tgtcaacagg cagtccaaca cccatcgctg tggctgccct 2820

actcattaca ctcttggaga tggcaataaa acttgtatag gtcctaggtc atttcttctg 2880

tatggtcaga ggaatacact gagtcgcctg ttacctgaca gtgatgactg ccctgatgtt 2940

gtgttgccta tacacagtgt caagaatgtc cgagctcttg aatatgatcc agtttcacag 3000

tacatttatt ggattgatgg tagagctcag tccatcaaga gatcatacga caacggcact 3060

catactggac cagcttttgt acctgctagc agtatccatt tcccatatga tatggcattg 3120

gatccatatg gaagagtact ttactggact tgtgcaaaca caaatgctat aaatgttaca 3180

agattggata atggatcagc tgttggtgtt gtggtcaagg tcgagggaga gaaacctcga 3240

aacattgtcc ttcatccaga aaaaggttta atgttttgga cagaagttgg tcaggcaccg 3300

cgcatcttac gagctcgaat ggatggcaaa tctcgtattg ttattgcttc agagattgag 3360

aatttgtctg ccctggcagt tgatagagtg gctgacttac tcttctgggc tcaacccaat 3420

cagattgaat cttcagatct gaatgggaaa ggaaggaaaa tactagtgag caaacatctg 3480

gttcaagtga cgagtttagc tgttcatgga gattatttgt attgggtcga caaagaccag 3540

caacaaattg aacgagtaaa taagacaaat ggtaacaatc gtacaaccat gctgctacga 3600

ttggctcacc tggtggatat tgtagctgta aattttccta gtgaaaagat gttctccagc 3660

cacggctgca gctcaagtca ccatcaaggt ggttgttccc atctgtgcat agcctcacca 3720

cctgcatcac ctactggctc cagtactatt cctgccacct gttcctgtcc acaaagtctg 3780

gtccttcatg aagatggacg aacatgtagt gcagttcctg catgtggacc agatctcttc 3840

acgtgtgctg ctccttcaaa ttccaacaaa gactgcatac caagcacctg gcgatgtgat 3900

ggacaggatg actgcccgga tgccagcgac gaagctggct gcccagattg taaaccagac 3960

gaattccgct gccagactgg acagtgtata gataaatctt gggtgtgtga cggaacttca 4020

cagtgtccag acaattatga cgagagcaga tgctgtgatt tccagtgtgc aagaactgga 4080

gtgtgtattc cacatactgc tgtatgtgac ggatatcaga actgtccaga tggcagtgac 4140

gaggcagatt cagaatgtag aggagccaat cctcgtgtag acgttatgtc atatggtgat 4200

tcaaacaaat ccacttacgt agtgagtatt ttggtcacta tagcatctgt tatcatgtta 4260

ggatctgttg gattctactg ccgtcgtcgt cttataagtt caaacaatgt aactttaaac 4320

gatgacctcg atgattcggc tggtgatcct ctgtcaccaa agccgataca tggtattgct 4380

aagcctatct gtccagtggc tggaatccaa aagaattgtc gactagacaa atgtaataag 4440

aaacctggta acgatgctgt tcgtatgtcg atgctgaatg gtagcacaac atccaacagt 4500

tatgacagga gtcatatcac aggtgcttca agctcaacca ctgaaggtgg tccaaatggt 4560

gctcctttac ttgtatcctc ttcttcgttg ctgtgctacc cccgcgagac tctgaatcct 4620

ccacccagtc cggctactac agcggactcg aatagaaaat tgcatcacaa ttataatgaa 4680

gactactgct gttcttcatc aggtgcatca agatatcgcc cctacagaca ttacagagcg 4740

atcaatcaac ccccaccacc cactccttgt agtacagatg tatgtgatga atctgattcg 4800

aactaccctc cgccctctag agggaggcag aggtattatg cacctcctgg ttcagaatat 4860

gattctgatc ctttcccacc tcctccaaca cctagatcgc actaccacag cgatggggga 4920

ggtgtgggag gattgccttc ttcttgtcca ccatcccctt catccacagc agggcgttct 4980

tcaacatact tcaatcctct tccaccgccg ccctcgcctg caccttcccc cggcagtcgc 5040

tgtgattgct ga 5052

<210> 3

<211> 3180

<212> DNA

<213> Periplaneta americana

<400> 3

atggaagaaa cgaaaatcat atatcatata gacgatgaag aaacacctta tttagttaaa 60

ctaactattg cgcctgaacg tgttacatta gcagacttca agaatgttct taatcgacca 120

aattacaagt tcttctttaa gtctatggac gatgattttg gtgttgtgaa ggaagagatt 180

attgacgacg atgctcatct accgtgtttc aatggaagag tagtgtcctg gttggtttct 240

gcagaaggca gcaacgtttc agatgggaca tcacagtgta ctgacagtgt agtgcattct 300

gatataaagg ttcctataga cagacctgga gatcctcaca ttattaatcc agtccatgga 360

ggtagaacaa atgtggagga cacaacatgc acggagacag aatctatcat cagctctcgc 420

caaggccatc ataatgttca caaaggatct agacctcatc atggagacaa gtacaataaa 480

tataatgggt tgcggataaa tggacactca aaacacagat ctgggcatgg ttatgaatca 540

agttcaatgc tcagctctga cttggaaacg acaagtttcc tggaatctga ggatgatgca 600

tcgagcagaa ttaccacaac cactggtaga gctaccaatc tttcagttga tagacagaca 660

ctgggaacag ctgaccatag cagtgtatca aggttgcact tatctggccg tcgtcgccca 720

cagcgccgtc gccgacatcg tgttccacct gccatgtctc gcacatcatc cttctcgtct 780

atcacagact ccaccatgtc gctcaacatc atcactgtta cacttaacat ggatactgtg 840

aactttcttg gtatcagcat agttggtcag agtaacaagg gtggagatgg tggtatctat 900

gttggatcta ttatgaaagg tggtgcagtt gctcttgatg ggcgcataga acctggtgat 960

atgatactac aagtgaatga tataaatttt gaaaacatgt ccaatgatga ggcagttcga 1020

gtgttaaggg aagttgttca gaaaccaggg cctatcaaac tagtggttgc aaaatgctgg 1080

gacccaaatc ctaaaggtta cttcacgatt cctcggactg aacctgtgag acccattgat 1140

ccaggagctt gggtggcaca cacggcagca attagaggag agtcgtttcc tgttcggcct 1200

ccatcagtaa caacccttac ctctaccagc tcctctatca catcgagtgt gcctgagaca 1260

gaacgaccat ttgaagagat tcagttgact gtaaacacag atatgccgac catcgttcga 1320

gccatggctc gacctgactc aggactagag attcgagatc gaatgtggtt aaagattaca 1380

ataccaaatg ctttcatagg gtgtgacgtt gtggattggc tacatgctca tgtagaaggt 1440

tttgtagatc gtcgagatgc tcgcaaatat gcttcacaga tgctgaaagc tggctacatc 1500

cgacatactg ttaataagat tacattctca gaacagtgtt actatgtttt tggagatctc 1560

tgtacggcaa tgtcaaatct gaaactccat gatgactgca catcgttaga tagggacaca 1620

gttggacctc ttcctgcacc aaacacaatg ccagcacctt ggggaggaac ccacatgcca 1680

tactcgggca cttacctacc tcatacaggt gcagggtatg ctcccatgcc atttaactac 1740

accaacgaac cttcagtgta tggatatgca cgtgaggaaa gtgtgcatag tggttcaggt 1800

ggctccagta atggttcaga gcctgtgtgt aaagaaggtg ctggagacct gatgaagtct 1860

ggcagctctg cgagtgagag cgagttggga ggtggtggaa acggtggtgt aggatcagct 1920

ataaaaccat ctggtggtcg acggtcgggc gggtccaggt cacgttctag tggctcagaa 1980

caatcggtag ctacagcctc gacagctgga ccacagtcac agcatccttt gcagcagacg 2040

caggatctat ccgttgaatt catatcttcc aagttcgaca gtcttagaga ggaattgcca 2100

catgcaagac atgaactcaa ctccacgcag gaggagatga agaggctagt gcaagaaaat 2160

gaccatctca aacaggaagt gagtgatctg cagcagtaca cacgcagaga caatataatg 2220

ctatttggag ttccggagat cgatgaacaa acaacttatg aagtcattga ccaaatatcg 2280

gaagtcatag gcggaagacg cgtcacaaag agtagaagtc gtgatgaatg gctccagctg 2340

ttcagaaatg aggccaaaaa tgatggcagc agtcctggga ttgcgacaaa gaaagtcaac 2400

agggaccttc cggcaggaag aatcacagca ggtgatcaac tgacagcagt gaccagagat 2460

cttttgatca agacaagggt tgcaagaaaa gatgaaggca gtcccaaatt tgaaattact 2520

ggttacggta ttgacgaaag agaagaagat aaggttgtgc tggtatctgg ggaacgcaga 2580

tatcatatcg catctgtaga cttcaacaac cgatatccag aaagaccacc aatttcaaat 2640

ttagagcaac tggaactgtt caaaagaagc caagtagcag ttgacacgct gtctatggta 2700

ttgcttgctc tcaactgcat tgttcgtgac caccgaaacc gaaatctgga ccactacgta 2760

aagaaaccga atgtaggtct ggcagagcag caacatcctg acggtagctt tggaaacctc 2820

cacagcacag ctctggcagt acaggcttta gaggctgcag atagtgaact tgctggcaat 2880

ggtggcaaca ttggcccagt gcactggaac cacagcagtg ctataaacta cctggtatca 2940

cgtcaggctc ctgatgattc gtttggagac gtgtttacaa ccactgaagt tgtgcttggc 3000

ttgggaccac gacgactcag ctctgttaga gacctcaatt gtggtagtga taattctgca 3060

ggaactttca ccgctagtgt agctaaactc catccgtcta cagcaacaac acttcctcct 3120

actggcaagt gcggattcaa atttgacagt ggaaggcatg actcctcgaa aaaatcctaa 3180

<210> 4

<211> 589

<212> DNA

<213> Artificial Sequence

<400> 4

taatacgact cactatagga gacggcgttc atatacgcag acggcgttca tatacgcgat 60

cacaagtgcg ggcgtgacac acgccatcgc gcgggcgtgc agcgagggca gcatcgagtc 120

gtgcacgtgt gattacagcc accaggcgcg ggcgccgcag gtgacgtccg tgcccggcct 180

gcgcgactgg gagtggggcg gctgctccga caacatcggc tacggcttca agttctcccg 240

cgaattcgtc gataccggcg agcgggggcg caacctccgc gagaagatga atctccacaa 300

caatgaggcc ggcagagcgc acgtttcctc ggagatgcgt caagaatgta agtgtcacgg 360

catgtctggc tcctgcacgg tcaagacctg ctggatgcgg ctgcccagct tccgagtcgt 420

aggcgacaac ctcaaggacc gcttcgacgg cgcctccaga gtgatggtga gtaacgcggg 480

cagcctgcgc ggccagggtg gtagcggcgg cagcggcgtc ggtggtaaga agaacagata 540

caacttccaa ctgaaaccct acaacccgga cctatagtga gtcgtatta 589

<210> 5

<211> 534

<212> DNA

<213> Artificial Sequence

<400> 5

taatacgact cactatagga acggcactca tactggacca gcttttgtac ctgctagcag 60

tatccatttc ccatatgata tggcattgga tccatatgga agagtacttt actggacttg 120

tgcaaacaca aatgctataa atgttacaag attggataat ggatcagctg ttggtgttgt 180

ggtcaaggtc gagggagaga aacctcgaaa cattgtcctt catccagaaa aaggtttaat 240

gttttggaca gaagttggtc aggcaccgcg catcttacga gctcgaatgg atggcaaatc 300

tcgtattgtt attgcttcag agattgagaa tttgtctgcc ctggcagttg atagagtggc 360

tgacttactc ttctgggctc aacccaatca gattgaatct tcagatctga atgggaaagg 420

aaggaaaata ctagtgagca aacatctggt tcaagtgacg agtttagctg ttcatggaga 480

ttatttgtat tgggtcgaca aagaccagca acaaacctat agtgagtcgt atta 534

<210> 6

<211> 522

<212> DNA

<213> Artificial Sequence

<400> 6

taatacgact cactatagga catgtccaat gatgaggcag ttcgagtgtt aagggaagtt 60

gttcagaaac cagggcctat caaactagtg gttgcaaaat gctgggaccc aaatcctaaa 120

ggttacttca cgattcctcg gactgaacct gtgagaccca ttgatccagg agcttgggtg 180

gcacacacgg cagcaattag aggagagtcg tttcctgttc ggcctccatc agtaacaacc 240

cttacctcta ccagctcctc tatcacatcg agtgtgcctg agacagaacg accatttgaa 300

gagattcagt tgactgtaaa cacagatatg ccgaccatcg ttcgagccat ggctcgacct 360

gactcaggac tagagattcg agatcgaatg tggttaaaga ttacaatacc aaatgctttc 420

atagggtgtg acgttgtgga ttggctacat gctcatgtag aaggttttgt agatcgtcga 480

gatgctcgca aatatgcttc acacctatag tgagtcgtat ta 522

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 7

taatacgact cactatagga gacggcgttc atatacgc 38

<210> 8

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 8

taatacgact cactataggt ccgggttgta gggtttca 38

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 9

ggatcctaat acgactcact ataggaacgg cactcatact ggacc 45

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 10

ggatcctaat acgactcact ataggtttgt tgctggtctt tgtcg 45

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 11

ggatcctaat acgactcact ataggacatg tccaatgatg aggca 45

<210> 12

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 12

ggatcctaat acgactcact ataggtgtga agcatatttg cgagc 45

<210> 13

<211> 532

<212> RNA

<213> Artificial Sequence

<400> 13

agacggcguu cauauacgcg aucacaagug cgggcgugac acacgccauc gcgcgggcgu 60

gcagcgaggg cagcaucgag ucgugcacgu gugauuacag ccaccaggcg cgggcgccgc 120

aggugacguc cgugcccggc cugcgcgacu gggagugggg cggcugcucc gacaacaucg 180

gcuacggcuu caaguucucc cgcgaauucg ucgauaccgg cgagcggggg cgcaaccucc 240

gcgagaagau gaaucuccac aacaaugagg ccggcagagc gcacguuucc ucggagaugc 300

gucaagaaug uaagugucac ggcaugucug gcuccugcac ggucaagacc ugcuggaugc 360

ggcugcccag cuuccgaguc guaggcgaca accucaagga ccgcuucgac ggcgccucca 420

gagugauggu gaguaacgcg ggcagccugc gcggccaggg ugguagcggc ggcagcggcg 480

ucggugguaa gaagaacaga uacaacuucc aacugaaacc cuacaacccg ga 532

<210> 14

<211> 496

<212> RNA

<213> Artificial Sequence

<400> 14

aacggcacuc auacuggacc agcuuuugua ccugcuagca guauccauuu cccauaugau 60

auggcauugg auccauaugg aagaguacuu uacuggacuu gugcaaacac aaaugcuaua 120

aauguuacaa gauuggauaa uggaucagcu guugguguug uggucaaggu cgagggagag 180

aaaccucgaa acauuguccu ucauccagaa aaagguuuaa uguuuuggac agaaguuggu 240

caggcaccgc gcaucuuacg agcucgaaug gauggcaaau cucguauugu uauugcuuca 300

gagauugaga auuugucugc ccuggcaguu gauagagugg cugacuuacu cuucugggcu 360

caacccaauc agauugaauc uucagaucug aaugggaaag gaaggaaaau acuagugagc 420

aaacaucugg uucaagugac gaguuuagcu guucauggag auuauuugua uugggucgac 480

aaagaccagc aacaaa 496

<210> 15

<211> 484

<212> RNA

<213> Artificial Sequence

<400> 15

acauguccaa ugaugaggca guucgagugu uaagggaagu uguucagaaa ccagggccua 60

ucaaacuagu gguugcaaaa ugcugggacc caaauccuaa agguuacuuc acgauuccuc 120

ggacugaacc ugugagaccc auugauccag gagcuugggu ggcacacacg gcagcaauua 180

gaggagaguc guuuccuguu cggccuccau caguaacaac ccuuaccucu accagcuccu 240

cuaucacauc gagugugccu gagacagaac gaccauuuga agagauucag uugacuguaa 300

acacagauau gccgaccauc guucgagcca uggcucgacc ugacucagga cuagagauuc 360

gagaucgaau gugguuaaag auuacaauac caaaugcuuu cauagggugu gacguugugg 420

auuggcuaca ugcucaugua gaagguuuug uagaucgucg agaugcucgc aaauaugcuu 480

caca 484

Claims (6)

1. American cockroach amputation regeneration associated genes, respectively two genes of Arr, Dsh, wherein the nucleotide sequence of Arr is such as Shown in SEQ ID No.2, the nucleotide sequence of Dsh is as shown in SEQ ID No.3.

2. the dsRNA of one group of targeted silent American cockroach amputation regeneration associated genes described in claim 1, the dsRNA have Two, difference two genes of targeted silent Arr, Dsh, wherein the nucleotide sequence such as SEQ of the dsRNA of targeted silent Arr gene Shown in ID No.14, the nucleotide sequence of the dsRNA of targeted silent Dsh gene is as shown in SEQ ID No.15.

3. one group of targeted silent American cockroach amputation regeneration associated genes described in claim 1 according to claim 2 DsRNA preparation method, which is characterized in that the dsRNA of targeted silent Arr gene is prepared by the following method: by SEQ ID Gene fragment clone shown in No.5 is named as pTOPO-Arr to pTOPO carrier, then using pTOPO-Arr as template, with two Primer SEQ ID No.9 and SEQ the ID No.10 containing T7 promoter is held, PCR amplification is carried out, PCR product passes through T7RiboMAX^TMExpress RNAi System transcription synthesis；The dsRNA of targeted silent Dsh gene is prepared by the following method: By gene fragment clone shown in SEQ ID No.6 to pTOPO carrier, it is named as pTOPO-Dsh, is then with pTOPO-Dsh Template contains primer SEQ ID No.11 and SEQ the ID No.12 of T7 promoter with both ends, carries out PCR amplification, and PCR product is logical Cross T7RiboMAX^TMExpress RNAi System transcription synthesis.

The nucleotide sequence of the American cockroach amputation regeneration associated genes of 4.dsRNA targeting is respectively such as SEQ ID No.5, SEQ ID Shown in No.6.

5. containing shown in SEQ ID No.5 or the recombinant vector or recombinant bacterium of nucleotide sequence shown in SEQ ID No.6.

6. one group of targeted silent American cockroach amputation regeneration associated genes described in claim 1 according to claim 2 DsRNA prevention and treatment American cockroach in application.