CN111936626B - Chitinase-like EN03 of diaphorina citri chitin binding protein, and encoding gene and application thereof - Google Patents
Chitinase-like EN03 of diaphorina citri chitin binding protein, and encoding gene and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
Abstract
The invention provides a diaphorina citri chitin binding protein Chitinase-like EN03, belonging to the technical field of pest control; the amino acid sequence of the diaphorina citri chitin binding protein Chitinase-like EN03 is shown as SEQ ID NO. 1. According to the invention, through RNA interference silencing of the encoding gene of the diaphorina citri chitin binding protein Chitinase-like EN03, the death caused by incomplete or unsuccessful molting of diaphorina citri wings can be realized, so that the effect of preventing and controlling diaphorina citri is achieved.
Description
Technical Field
The invention relates to the technical field of pest control, in particular to a diaphorina citri chitin binding protein Chitinase-like EN03, and a coding gene and application thereof.
Background
The Diaphorina citri (Diaphorina citri) belongs to the family of Diaphorina, the family of Diaphorina citri, is a main pest in the tender tip stage of citrus, and is the only insect vector carrier for natural transmission of citrus yellow-shoot bacteria found by current research. Citrus yellow long disease (Citrus Huanglongbing, HLB) is the most devastating disease of the world citrus industry, causing huge economic loss to the citrus industry. The strict control of diaphorina citri is a key measure for the current control of citrus yellow long disease. The current control of diaphorina citri is mainly chemical control and biological control, namely, the diaphorina citri is killed by chemical pesticides or natural enemy insects and entomopathogenic fungi. No report on the structure and function of key genes of the diaphorina citri is found.
Disclosure of Invention
The invention aims to provide a diaphorina citri chitin binding protein Chitinase-like EN03, and a coding gene and application thereof, wherein the diaphorina citri chitin binding protein Chitinase-like EN03 and the coding gene thereof can be used for preventing and controlling diaphorina citri.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a diaphorina citri chitin binding protein Chitinase-like EN03, and the amino acid sequence is shown as SEQ ID NO. 1.
The invention also provides a coding gene of the diaphorina citri chitin binding protein Chitinase-like EN03, and the nucleotide sequence is shown as SEQ ID NO. 2.
The invention also provides the dsRNA of the coding gene in the scheme, and the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 3.
The invention also provides application of the diaphorina citri chitin binding protein Chitinase-like EN03 or the coding gene or the dsRNA in the prevention and treatment of diaphorina citri.
Preferably, the application comprises the silencing of the gene encoding Chitinase-like EN03 by RNA interference to control diaphorina citri.
Preferably, the application comprises the steps of: placing the diaphorina citri larvae into the dsRNA aqueous solution for soaking for 3-10 min; the concentration of dsRNA in the dsRNA aqueous solution is 50-300 ng/. Mu.L; the solvent of the dsRNA aqueous solution is enzyme-free water.
Preferably, the diaphorina citri larvae comprise 4-5-year-old diaphorina citri larvae.
Preferably, the diaphorina citri larvae are starved for 1.5-2.5 hours before soaking.
The invention has the beneficial effects that: the invention provides a diaphorina citri chitin binding protein Chitinase-like EN03, and the amino acid sequence is shown as SEQ ID NO. 1. According to the invention, through RNA interference silencing of the encoding gene of the diaphorina citri chitin binding protein Chitinase-like EN03, the diaphorina citri wing can be subjected to incomplete or unsuccessful molting, so that death is caused, and the effect of preventing and controlling diaphorina citri is achieved.
Drawings
FIG. 1 is a schematic representation of screening for diaphorina citri chitin binding proteins;
FIG. 2 shows the extraction and ultrafiltration of diaphorina citri epidermal proteins to remove SDS; wherein lane 1: marker, lanes 2-3: PBS washes 1, 2, 3 times, lanes 4-6: wash with 2% sds 1, 2, 3 times, lane 7: mixed solution of 1 st, 2 nd and 3 rd times washed with 2% sds, lane 8: protein solution after KCl removal of SDS, lane 9: ultrafiltering to remove the protein solution after SDS;
FIG. 3 is a chitin-binding protein screening experiment of protein solution after SDS removal; lane 1: marker, lane 2: protein solution after SDS removal, lane 3: effluent, lanes 4-6: wash buffer washes 1, 2, 3 times, lane 7: eluting with 8M urea;
FIG. 4 is a mass spectrum of Chitinase-like ENO 3;
FIG. 5 shows the results of an in vitro chitin binding assay for prokaryotic expressed proteins;
FIG. 6 shows the change in the expression level of DcCHT-ENO3 gene after RNAi;
FIG. 7 shows the change in the diaphorina citri emergence phenotype after silencing the DcCHT-ENO3 gene.
Detailed Description
The invention provides a diaphorina citri chitin binding protein Chitinase-like EN03, the amino acid sequence of which is shown as SEQ ID NO.1, which specifically comprises the following steps: MRALSVLLFVAFAIYYCEGVAKTVCYYNHKAFKRDGTAKVGPEELKPALSMCTHLVYGFAGISDSGDYHIKSLDKELDTDKNKGKELFKQITALKTFQPNLNIMLSVGGFEDDDDKEKYLEVLDDPKYRKSFIETTVAALKKYGFNGLDLAWEFPVVTEKHESYTLGSIWHKIKKTVTGPKDDNPTLHREHFTLLIREMKAAFRAENFLLSASVLPHVNYTVYFDVPSITQHLDMITLHAYDFRTPQRNPKEADYSAPLHFVYGRVPHQNANAMVRWFIEHGVELQKLVLGIPTFGRSWLLEESSHKSGIPPLVADGAGEKGTITKEEGLLSYAEICPQLVSITNAQASPSLLRKQEDPQRRLGTYAFRLADKKLKQEYGTWVSFEEPETAGYKASYAKLAGLGGVAIIDLSLDDFRGMCNSNKFPILRAARTQFV.
The invention also provides a coding gene of the diaphorina citri chitin binding protein Chitinase-like EN03, which has a nucleotide sequence shown in SEQ ID NO.2, and specifically comprises the following steps: atgcgagctctaagtgttcttttgttcgtcgcttttgcgatatactactgtgaaggcgtggccaaaacggtgtgctactacaaccataaggcattcaagagagatggaacggccaaagttggacccgaggagctgaagccagccctgagcatgtgcacccatttggtgtacgggttcgctgggatttcggacagcggcgactaccacatcaagtccctggacaaggaattggacacggacaagaacaaaggcaaagagctgttcaaacagataaccgcgctgaaaactttccaacccaacctgaacattatgcttagtgtgggaggatttgaagatgatgatgacaaggagaagtatttggaagtgctggatgacccgaaatacagaaagagtttcatcgaaacaactgtggcagctctgaaaaagtacggcttcaacggattggatctggcctgggaattccccgttgtgactgaaaagcacgaatcttacactcttggatccatctggcataaaatcaagaaaaccgtcaccggacccaaggatgacaatccaacccttcatcgtgaacatttcactctgctcatccgagaaatgaaagccgccttccgagctgagaacttcctactgagtgcctccgttttgccgcacgtcaattataccgtatactttgacgtcccctccatcactcaacatctggacatgatcactctccatgcctatgacttccgtacaccccagagaaaccccaaggaggctgactattccgctccccttcactttgtctacggtagagtacctcatcagaatgctaacgctatggtgcgctggttcattgaacatggagttgaattacaaaaactggtccttggaatccccaccttcggtcgctcttggcttctggaggagtcaagtcacaaatccggcatccctccccttgtagctgatggggccggagaaaagggcaccatcaccaaagaagaaggtctcctctcatacgccgaaatctgtcctcaactggtcagcattaccaatgcgcaagcaagccccagtctactgcgcaaacaagaagatcctcagagaagattaggaacgtacgcattcagattagcagacaagaagttgaagcaggaatatggtacttgggttagttttgaggaacccgagacggctggatacaaagcgtcctatgccaagctggcaggtttgggaggagtggctatcattgatctgagtctggatgacttccgaggcatgtgcaattccaacaagttccccatattgagagccgctagaacacagtttgtgtag.
The invention also provides the dsRNA of the coding gene, which has the nucleotide sequence shown in SEQ ID NO.3, and specifically comprises the following steps: tgttcttttgttcgtcgcttttgcgatatactactgtgaaggcgtggccaaaacggtgtgctactacaaccataaggcattcaagagagatggaacggccaaagttggacccgaggagctgaagccagccctgagcatgtgcacccatttggtgtacgggttcgctgggatttcggacagcggcgactaccacatcaagtccctggacaaggaattggacacggacaagaacaaaggcaaagagctgttcaaacagataaccgcgctgaaaactttccaacccaacctgaacattatgcttagtgtgggaggatttgaagatgatgatgacaaggagaagtatttggaagtgctggatgacccgaaatacagaaagagtttcatcgaaacaactgtggcagctctgaaaaagtacggcttcaacggattggatctggcctgggaattccccgttgtgactgaaaagcacgaat.
The invention also provides an application of the diaphorina citri chitin binding protein Chitinase-like EN03 (DcCHT-ENO 3) or the coding gene or the dsRNA in the prevention and treatment of diaphorina citri; the invention preferably silence coding genes of the Chitinase-like EN03 through RNA interference to prevent and treat diaphorina citri; alternatively, protein inhibitors of the diaphorina citri chitin binding protein are used to control diaphorina citri; the protein inhibitor preferably comprises alloxacin.
According to the invention, through RNA interference silencing of the encoding gene of the diaphorina citri chitin binding protein Chitinase-like EN03, the death caused by incomplete or unsuccessful molting of diaphorina citri wings can be realized, so that the effect of preventing and controlling diaphorina citri is achieved. Incomplete molting means that part of epidermis such as wings, feet and the like is also connected with the front epidermis, if the wings and the feet are also connected with the front epidermis, a series of activities such as flying, jumping and the like of the diaphorina citri can be influenced, feeding (phenomenon that death can be caused by feeding incapacity) of the diaphorina citri is influenced to a certain extent, mating oviposition (reproduction can be caused by mating incapacity) and the like are influenced, and therefore, the control of the diaphorina citri is achieved.
In the present invention, the application of the dsRNA in controlling diaphorina citri preferably includes: placing the diaphorina citri larvae into the dsRNA aqueous solution for soaking for 3-10 min, preferably 5-8 min; the concentration of dsRNA in the aqueous dsRNA solution is preferably 50-300 ng/. Mu.L, more preferably 100-200 ng/. Mu.L, and most preferably 150 ng/. Mu.L; the solvent of the aqueous dsRNA solution is preferably enzyme-free water; the diaphorina citri larvae preferably comprise 4-5-year-old diaphorina citri larvae; the diaphorina citri larvae preferably further comprise starvation treatment for 1.5-2.5 hours, preferably 2 hours, before soaking.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 screening of diaphorina citri chitin binding proteins (schematic view see fig. 1), the specific procedure is as follows:
1. extraction of total protein of diaphorina citri epidermis
(1) Taking the skin of the adult diaphorina citri in a 2mL centrifuge tube, and washing twice with PBS (in the process of taking the skin of the diaphorina citri, the taken skin needs to be immediately placed on ice);
(2) Rapidly putting the sample obtained in the step (1) into an ultralow temperature refrigerator (-80 ℃) for freezing;
(3) Taking out the sample in the step (2) from the low-temperature refrigerator, and grinding the liquid nitrogen into powder;
(4) Adding precooled PBS according to the proportion of 0.5 g/mL;
(5) Fully and uniformly mixing, and centrifuging at 12000rpm for 10min at 4 ℃, and taking supernatant to obtain PBS-1;
(6) Repeating the steps (4) and (5) twice to obtain PBS-2 and PBS-3 respectively;
(7) Recovering the precipitate in the step (6), adding 2% SDS extraction buffer solution according to the proportion of 0.5g/mL, and placing the centrifuge tube on a horizontal shaking table for shaking at a medium speed for 1h;
(8) Centrifuging at 12000rpm for 10min at 4deg.C, collecting supernatant to obtain SDS-1;
(9) Repeating the steps (7) and (8) twice to obtain SDS-2 and SDS-3 respectively;
(10) Detecting the protein extraction condition by SDS-PAGE, wherein the detection result is shown in figure 2, and figure 2 shows that the protein of the diaphorina citri epidermis is extracted and the SDS is removed by ultrafiltration; wherein lane 1: marker, lanes 2-3: PBS washes 1, 2, 3 times, lanes 4-6: wash with 2% sds 1, 2, 3 times, lane 7: mixed solution of 1 st, 2 nd and 3 rd times washed with 2% sds, lane 8: protein solution after KCl removal of SDS, lane 9: the protein solution after SDS was removed by ultrafiltration.
2. Ultrafiltration to remove SDS
(1) Mixing SDS-1, SDS-2 and SDS-3 obtained by the experiment to obtain an S solution;
(2) Adding 1M KCl (200 mu L of 1M KCl is added per milliliter of S solution) into the S solution, and fully and uniformly mixing;
(3) Centrifuging at 12000rpm at 4deg.C for 10min, and collecting supernatant;
(4) Then the solution obtained in (3) was diluted 20-fold with 1mM HEPES, and added to a 10K ultrafilter tube;
(5) Centrifuging at 7830rpm at 25deg.C for 20min, collecting inner tube solution in 1.5mL centrifuge tube, and storing at-20deg.C.
3. Treatment of proteins of interest with chitin and cellulose
(1) Treatment of chitin and cellulose:
a. taking 1mL of chitin beads, adding 6 times of column volume of binding buffer (20mM HEPES,15mM NaCl,pH7.4), covering a cover, shaking on a horizontal shaking table for 1h, and storing at 4deg.C for later use;
b. taking a proper amount of cellulose powder, placing the cellulose powder in a binding buffer solution for soaking overnight, taking 1mL of the cellulose powder, placing the cellulose powder in a chromatographic column, and placing the chromatographic column at 4 ℃ for standby.
(2) Taking a protein solution from which SDS is removed, adding an equal volume of binding buffer solution into the protein solution, and fully and uniformly mixing;
(3) Adding the solution obtained in the step (2) into a chromatographic column containing chitin (cellulose), placing the chromatographic column on a horizontal shaking table for medium-speed incubation for 2 hours, and collecting effluent FT;
(4) Adding 5mL of washing buffer (20mM HEPES,1M NaCl,pH7.4) into the chromatographic column for rinsing, placing the chromatographic column on a horizontal shaking table, shaking at a medium speed for 15min, and collecting washing liquid W1;
(5) Repeating the step (4) twice, and respectively collecting washing solutions W2 and W3;
(6) Adding 2mL of 8M urea into a chromatographic column, placing the chromatographic column on a horizontal shaking table, incubating at 25 ℃ for 1h, and collecting eluent E;
(7) SDS-PAGE analysis, the results of which are shown in FIG. 3, and FIG. 3 shows a chitin binding protein screening experiment of the protein solution after SDS removal; lane 1: marker, lane 2: protein solution after SDS removal, lane 3: effluent, lanes 4-6: wash buffer washes 1, 2, 3 times, lane 7:8M urea elution.
4. LC-MS/MS mass spectrometry
The protein bands were sent to Shanghai Bioengineering Co.Ltd for LC-MS/MS analysis, see FIG. 4 for analysis results. As can be seen from fig. 4, the peptide fragments obtained by mass spectrometry are compared in the diaphorina citri genome database, and the comparison result is the Chitinase-like EN03 gene.
Example 2 chitin in vitro binding experiments of prokaryotic expression proteins
Prokaryotic expression of the coding gene of Chitinase-like EN03 is entrusted to the Hangzhou Hua' an biological company, and the obtained purified protein is subjected to an in vitro chitin binding experiment, and the experimental method is referred to step 3 in example 1, and only the final concentration of NaCl in the binding buffer is required to be increased to 500mM. The results are shown in fig. 5, where M: protein marker, FT: effluent, W1-3: washing liquid first, second and third times, E: and (3) eluting the eluent. From FIG. 5, it can be seen that the Chitinase-like EN03 protein obtained by prokaryotic expression can bind chitin in vitro and has chitin binding activity. Cellulose as a control can also be incorporated.
EXAMPLE 3 Synthesis of dsRNA and RNA interference analysis
1. Synthesis of dsRNA
According to the Chitinase-like EN03 protein sequence, the corresponding gene sequence (> XM_026833073.1PREDICTED:Diaphorina citri Chitinase-like protein EN03 (LOC 103523836), mRNA and nucleotide sequence shown in SEQ ID NO. 4: tcgaatcaacgcttggctccgggtttattcaaattgagttctgattgcaagatgcgagctctaagtgttcttttgttcgtcgcttttgcgatatactactgtgaaggcgtggccaaaacggtgtgctactacaaccataaggcattcaagagagatggaacggccaaagttggacccgaggagctgaagccagccctgagcatgtgcacccatttggtgtacgggttcgctgggatttcggacagcggcgactaccacatcaagtccctggacaaggaattggacacggacaagaacaaaggcaaagagctgttcaaacagataaccgcgctgaaaactttccaacccaacctgaacattatgcttagtgtgggaggatttgaagatgatgatgacaaggagaagtatttggaagtgctggatgacccgaaatacagaaagagtttcatcgaaacaactgtggcagctctgaaaaagtacggcttcaacggattggatctggcctgggaattccccgttgtgactgaaaagcacgaatcttacactcttggatccatctggcataaaatcaagaaaaccgtcaccggacccaaggatgacaatccaacccttcatcgtgaacatttcactctgctcatccgagaaatgaaagccgccttccgagctgagaacttcctactgagtgcctccgttttgccgcacgtcaattataccgtatactttgacgtcccctccatcactcaacatctggacatgatcactctccatgcctatgacttccgtacaccccagagaaaccccaaggaggctgactattccgctccccttcactttgtctacggtagagtacctcatcagaatgctaacgctatggtgcgctggttcattgaacatggagttgaattacaaaaactggtccttggaatccccaccttcggtcgctcttggcttctggaggagtcaagtcacaaatccggcatccctccccttgtagctgatggggccggagaaaagggcaccatcaccaaagaagaaggtctcctctcatacgccgaaatctgtcctcaactggtcagcattaccaatgcgcaagcaagccccagtctactgcgcaaacaagaagatcctcagagaagattaggaacgtacgcattcagattagcagacaagaagttgaagcaggaatatggtacttgggttagttttgaggaacccgagacggctggatacaaagcgtcctatgccaagctggcaggtttgggaggagtggctatcattgatctgagtctggatgacttccgaggcatgtgcaattccaacaagttccccatattgagagccgctagaacacagtttgtgtagatttctgcgggtatttacttagggtgtgaaagcagtctgttttttcatttaatttcgagcaatatgtctgtgggtattattttacgaatatgtataatatttccgtgtcataggtatttcagttactggagcataaaatcaaactattaagcag) is found in the NCBI database, and a specific Primer is designed by using Primer premier5.0 software, and all primers are synthesized by Shanghai bioengineering Co. Taking 5-10 pieces of extracted RNA of the adult diaphorina citri (the method step refers to a Simmen RNA extraction kit), and reversely transcribing the extracted RNA into cDNA (the method step refers to a Simmen reverse transcription kit). Amplifying the open reading frame (the nucleotide sequence is shown as SEQ ID NO. 2) of the gene by using a specific primer and a PCR technology by using cDNA as a template, and recovering an amplified product by using an Omega gel recovery kit; the amplified product is connected into PMD-19T plasmid, transformed into colibacillus, plated, single colony is picked on 10 mu L of LB liquid culture medium the next day, 2 mu L of template for colony PCR, 8 mu L of ampicillin liquid culture medium for shaking, agarose gel electrophoresis is carried out on the product of colony PCR, and the bacterial solution with the target band is sent to sequence; selecting bacterial liquid extracting plasmid with lowest mutation rate (using a rapid small plasmid extracting kit according to the method steps in the specification); using plasmid as template, using primer containing T7 promoter to make PCR amplification, recovering amplification product gel; the concentration of the recovered product was measured, and dsRNA was synthesized by using Promega T7 RiboMAX Express RNAi System (method steps in the specification), and the nucleotide sequence of the synthesized dsRNA was shown in SEQ ID NO. 3.
(2) RNAi analysis
Diluting the synthesized dsRNA to 50, 150 and 300 ng/mu L respectively by using enzyme-free water, and placing the dsRNA on ice for standby; selecting 4-5-year nymphs of diaphorina citri on a grafting film, dripping 5 mu L dsRNA on each grafting film, soaking for five minutes, and then placing the grafting film on the Murraya koenigii branches for feeding; each group of 30 nymphs, with the corresponding concentration of dsGFP as a control; five nymphs were taken after 48h and 72h, RNA was extracted, reverse transcribed into cDNA, and qPCR was performed to detect changes in the expression level. Primers used for qPCR: f:5'-GGATGACCCGAAATACAGAAAGA-3', as shown in SEQ ID NO. 5; r:5'-AACGGAGGCACTCAGTAGGAAG-3', as shown in SEQ ID NO. 6; reaction System ddH 2 O8. Mu.L, SYBR 10. Mu.L, upstream and downstream primers 0.5. Mu.L, cDNA template 1. Mu.L, 20. Mu.L overall; the reaction procedure: the reaction condition is that the pre-denaturation is carried out for 10min at 95 ℃;95℃10s,60℃10s,72℃10s,45 cycles. Experiments were repeated 6 times with 6 independent samples and the reactions were all completed on a Roche LightCycler480 II real-time quantitative PCR instrument. Phenotypic changes in nymph emergence were observed (results shown in fig. 6 and 7). FIGS. 6 and 7 are RNAi results of the diaphorina citri chitin binding protein Chitinase-like protein ENO 3; wherein FIG. 6 shows the change in the expression level of DcCHT-ENO3 gene after RNAi. Using dsGFP as a control, the relative expression levels of the CHT-ENO3 genes at 48h and 72h after dsDcCHT-ENO3 treatment were significantly down-regulated compared to the control group, indicating successful silencing of the DcCHT-ENO3 gene. FIG. 7 shows the change in the diaphorina citri emergence phenotype after silencing the DcCHT-ENO3 gene. The control group showed normal emergence of diaphorina citri on the left, the experimental group showed abnormal emergence of diaphorina citri on the right, the appearance of incomplete molting on the diaphorina citri wings, and the death of the diaphorina citri wings due to unsuccessful molting.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Gannan university of teachers and students
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<213> Diaphorina citri (Diaphorina citri)
<400> 2
atgcgagctc taagtgttct tttgttcgtc gcttttgcga tatactactg tgaaggcgtg 60
gccaaaacgg tgtgctacta caaccataag gcattcaaga gagatggaac ggccaaagtt 120
ggacccgagg agctgaagcc agccctgagc atgtgcaccc atttggtgta cgggttcgct 180
gggatttcgg acagcggcga ctaccacatc aagtccctgg acaaggaatt ggacacggac 240
aagaacaaag gcaaagagct gttcaaacag ataaccgcgc tgaaaacttt ccaacccaac 300
ctgaacatta tgcttagtgt gggaggattt gaagatgatg atgacaagga gaagtatttg 360
gaagtgctgg atgacccgaa atacagaaag agtttcatcg aaacaactgt ggcagctctg 420
aaaaagtacg gcttcaacgg attggatctg gcctgggaat tccccgttgt gactgaaaag 480
cacgaatctt acactcttgg atccatctgg cataaaatca agaaaaccgt caccggaccc 540
aaggatgaca atccaaccct tcatcgtgaa catttcactc tgctcatccg agaaatgaaa 600
gccgccttcc gagctgagaa cttcctactg agtgcctccg ttttgccgca cgtcaattat 660
accgtatact ttgacgtccc ctccatcact caacatctgg acatgatcac tctccatgcc 720
tatgacttcc gtacacccca gagaaacccc aaggaggctg actattccgc tccccttcac 780
tttgtctacg gtagagtacc tcatcagaat gctaacgcta tggtgcgctg gttcattgaa 840
catggagttg aattacaaaa actggtcctt ggaatcccca ccttcggtcg ctcttggctt 900
ctggaggagt caagtcacaa atccggcatc cctccccttg tagctgatgg ggccggagaa 960
aagggcacca tcaccaaaga agaaggtctc ctctcatacg ccgaaatctg tcctcaactg 1020
gtcagcatta ccaatgcgca agcaagcccc agtctactgc gcaaacaaga agatcctcag 1080
agaagattag gaacgtacgc attcagatta gcagacaaga agttgaagca ggaatatggt 1140
acttgggtta gttttgagga acccgagacg gctggataca aagcgtccta tgccaagctg 1200
gcaggtttgg gaggagtggc tatcattgat ctgagtctgg atgacttccg aggcatgtgc 1260
aattccaaca agttccccat attgagagcc gctagaacac agtttgtgta g 1311
<210> 3
<211> 473
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tgttcttttg ttcgtcgctt ttgcgatata ctactgtgaa ggcgtggcca aaacggtgtg 60
ctactacaac cataaggcat tcaagagaga tggaacggcc aaagttggac ccgaggagct 120
gaagccagcc ctgagcatgt gcacccattt ggtgtacggg ttcgctggga tttcggacag 180
cggcgactac cacatcaagt ccctggacaa ggaattggac acggacaaga acaaaggcaa 240
agagctgttc aaacagataa ccgcgctgaa aactttccaa cccaacctga acattatgct 300
tagtgtggga ggatttgaag atgatgatga caaggagaag tatttggaag tgctggatga 360
cccgaaatac agaaagagtt tcatcgaaac aactgtggca gctctgaaaa agtacggctt 420
caacggattg gatctggcct gggaattccc cgttgtgact gaaaagcacg aat 473
<210> 4
<211> 1516
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tcgaatcaac gcttggctcc gggtttattc aaattgagtt ctgattgcaa gatgcgagct 60
ctaagtgttc ttttgttcgt cgcttttgcg atatactact gtgaaggcgt ggccaaaacg 120
gtgtgctact acaaccataa ggcattcaag agagatggaa cggccaaagt tggacccgag 180
gagctgaagc cagccctgag catgtgcacc catttggtgt acgggttcgc tgggatttcg 240
gacagcggcg actaccacat caagtccctg gacaaggaat tggacacgga caagaacaaa 300
ggcaaagagc tgttcaaaca gataaccgcg ctgaaaactt tccaacccaa cctgaacatt 360
atgcttagtg tgggaggatt tgaagatgat gatgacaagg agaagtattt ggaagtgctg 420
gatgacccga aatacagaaa gagtttcatc gaaacaactg tggcagctct gaaaaagtac 480
ggcttcaacg gattggatct ggcctgggaa ttccccgttg tgactgaaaa gcacgaatct 540
tacactcttg gatccatctg gcataaaatc aagaaaaccg tcaccggacc caaggatgac 600
aatccaaccc ttcatcgtga acatttcact ctgctcatcc gagaaatgaa agccgccttc 660
cgagctgaga acttcctact gagtgcctcc gttttgccgc acgtcaatta taccgtatac 720
tttgacgtcc cctccatcac tcaacatctg gacatgatca ctctccatgc ctatgacttc 780
cgtacacccc agagaaaccc caaggaggct gactattccg ctccccttca ctttgtctac 840
ggtagagtac ctcatcagaa tgctaacgct atggtgcgct ggttcattga acatggagtt 900
gaattacaaa aactggtcct tggaatcccc accttcggtc gctcttggct tctggaggag 960
tcaagtcaca aatccggcat ccctcccctt gtagctgatg gggccggaga aaagggcacc 1020
atcaccaaag aagaaggtct cctctcatac gccgaaatct gtcctcaact ggtcagcatt 1080
accaatgcgc aagcaagccc cagtctactg cgcaaacaag aagatcctca gagaagatta 1140
ggaacgtacg cattcagatt agcagacaag aagttgaagc aggaatatgg tacttgggtt 1200
agttttgagg aacccgagac ggctggatac aaagcgtcct atgccaagct ggcaggtttg 1260
ggaggagtgg ctatcattga tctgagtctg gatgacttcc gaggcatgtg caattccaac 1320
aagttcccca tattgagagc cgctagaaca cagtttgtgt agatttctgc gggtatttac 1380
ttagggtgtg aaagcagtct gttttttcat ttaatttcga gcaatatgtc tgtgggtatt 1440
attttacgaa tatgtataat atttccgtgt cataggtatt tcagttactg gagcataaaa 1500
tcaaactatt aagcag 1516
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ggatgacccg aaatacagaa aga 23
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aacggaggca ctcagtagga ag 22
Claims (3)
1. Application of dsRNA of encoding gene of diaphorina citri chitin binding protein Chitinase-likeeN03 in controlling diaphorina citri;
the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2;
the nucleotide sequence of the dsRNA is shown as SEQ ID NO. 3;
the application comprises the following steps:
placing the diaphorina citri larvae into the dsRNA aqueous solution for soaking for 3-10 min; the concentration of dsRNA in the dsRNA aqueous solution is 50-300 ng/. Mu.L; the solvent of the dsRNA aqueous solution is enzyme-free water.
2. The use according to claim 1, wherein the diaphorina citri larvae comprise 4-5 instar diaphorina citri larvae.
3. The use according to claim 1, wherein the diaphorina citri larvae are starved for 1.5-2.5 hours prior to soaking.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2020/100775 WO2022006764A1 (en) | 2020-07-08 | 2020-07-08 | Recombinant diaphorina citri chitinase-like protein en03, and coding gene and application thereof |
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| CN111936626A CN111936626A (en) | 2020-11-13 |
| CN111936626B true CN111936626B (en) | 2023-10-17 |
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| WO (1) | WO2022006764A1 (en) |
| ZA (1) | ZA202210581B (en) |
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| CN113444152B (en) * | 2021-07-05 | 2022-02-01 | 湖北省生物农药工程研究中心 | Protein BVP10 for preventing and treating spider mites and application thereof |
| CN114752610B (en) * | 2022-05-07 | 2023-08-11 | 赣南师范大学 | Application of diaphorina citri ubiquitin conjugated enzyme E2J2 gene in prevention and control of citrus yellow dragon disease |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101370941A (en) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | dsRNA as insect control agent |
| CN106413381A (en) * | 2014-06-06 | 2017-02-15 | 康奈尔大学 | Compositions and methods for deterring feeding by psyllids |
| CN107828792A (en) * | 2017-11-30 | 2018-03-23 | 大连理工大学 | Insect I types chitin-binding protein AA10 genes and its application in control of insect |
| CN109588562A (en) * | 2004-04-09 | 2019-04-09 | 孟山都技术有限公司 | For controlling the composition and method of insect infestations in plant |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR102017001164A2 (en) * | 2016-01-26 | 2019-03-06 | Embrapa - Empresa Brasileira De Pesquisa Agropecuária | DOUBLE TAPE RNA COMPOSITIONS FOR CITRI DIAPHORINE CONTROL AND METHODS OF USE. |
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2020
- 2020-07-08 WO PCT/CN2020/100775 patent/WO2022006764A1/en active Application Filing
- 2020-07-08 CN CN202080001360.7A patent/CN111936626B/en active Active
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109588562A (en) * | 2004-04-09 | 2019-04-09 | 孟山都技术有限公司 | For controlling the composition and method of insect infestations in plant |
| CN101370941A (en) * | 2006-01-12 | 2009-02-18 | 德福根有限公司 | dsRNA as insect control agent |
| CN106413381A (en) * | 2014-06-06 | 2017-02-15 | 康奈尔大学 | Compositions and methods for deterring feeding by psyllids |
| CN107828792A (en) * | 2017-11-30 | 2018-03-23 | 大连理工大学 | Insect I types chitin-binding protein AA10 genes and its application in control of insect |
Non-Patent Citations (5)
| Title |
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| GenBank.XM_026833073.1.《NCBI》.2018, * |
| GenPept.XP_026688874.1.《NCBI》.2018, * |
| Silencing Chitinase Genes Increases Susceptibility of Tetranychus cinnabarinus (Boisduval) to Scopoletin;Hong Zhou等;《BioMed Research International》;20171231;文献号9579736，第1-13页 * |
| XM_026833073.1;GenBank;《NCBI》;20181022;全文，尤其是ORIGIN部分 * |
| XP_026688874.1;GenPept;《NCBI》;20181022;全文，尤其是ORIGIN部分 * |
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| CN111936626A (en) | 2020-11-13 |
| ZA202210581B (en) | 2022-12-21 |
| WO2022006764A1 (en) | 2022-01-13 |
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