CN105400807A - Gypsy moth beta-N-acetylglucosamine glycosidase gene based on gene silencing technology - Google Patents

Gypsy moth beta-N-acetylglucosamine glycosidase gene based on gene silencing technology Download PDF

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CN105400807A
CN105400807A CN201510974582.8A CN201510974582A CN105400807A CN 105400807 A CN105400807 A CN 105400807A CN 201510974582 A CN201510974582 A CN 201510974582A CN 105400807 A CN105400807 A CN 105400807A
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dsrna
gene
gypsymoth
acetylglucosamine
beta
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CN105400807B (en
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范晓军
张建栋
杨春
刘建红
李瑶
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Taiyuan University of Technology
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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)
    • 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
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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)
    • C12Y302/0105—Alpha-N-acetylglucosaminidase (3.2.1.50)

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the field of agricultural biotechnology, in particular to a gypsy moth beta-N-acetylglucosamine glycosidase gene based on the gene silencing technology, a lethal gene fragment of the gypsy moth beta-N-acetylglucosamine glycosidase gene and dsRNA of the lethal gene fragment, and application of the dsRNA to killing pests. The lethal gene fragment LdNag2 and the dsRNA of the lethal gene fragment LdNag2 are acquired according to the gypsy moth beta-N-acetylglucosamine glycosidase gene LdNag1, the purpose of controlling pests is achieved through the dsRNA, it is hopeful to reduce the phenomenon that a large amount of pesticide is used, the environment is protected, production cost is reduced, economic benefits are increased, the gypsy moth beta-N-acetylglucosamine glycosidase gene has market application prospects, and a new way is provided for developing a safe and pollution-free pest control method.

Description

Based on the gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes of gene silent technology
Technical field
The present invention relates to agricultural biological technical field, specifically the application of dsRNA and dsRNA in lethal insect of a kind of β-N-Acetyl-D-glucosamine glycosidase genes of the gypsymoth based on gene silent technology and lethal gene fragment wherein and this lethal gene fragment.
Background technology
In China, the continuous single life-time service of chemical agent has caused insect to create resistance in various degree to Multiple Pesticides, needs to continue to increase Pesticide use amount and just can reach satisfied prevention effect, cause even more serious environmental pollution, forms vicious cycle.Gypsymoth also can affect human health in addition, and when the direct dirty dancing poison moth of people, redness often can occur and to itch phenomenon, severe patient even produces fash.Therefore, in agricultural production practice, be badly in need of the alternative means of prevention outside chemical pesticide.
RNA disturbs (RNAinterference, RNAi) be a kind of gene silencing phenomenon by double chain RNA mediate, the final processed size of double-stranded RNA is about the tiny RNA (siRNA) of 22nt, the mode of being matched by sequence is combined with protein coding gene, according to the degree degraded target gene mRNA of sequence pairing or the protein translation process of suppressor gene.RNAi is present in fungi, plant and animal widely.Within 2006, AndreFire and CraigMello is because finding that RNAi phenomenon is awarded Nobel's medical science and physiology is encouraged.
In organism, it is necessary that some important gene pairss sustain life.Therefore, theoretically, if utilize RNA perturbation technique the expression of important gene in Agricultural pests to be disturbed, then can cause the teratogenesis of insect or lethal, thus reach the object of Control pests.
Chitin degrading and metabolism are distinctive biological phenomenas in insect constant pitch main drive object, and owing to not containing chitin in the mankind and other higher animal bodies, therefore insect chitin degeneration system has been acknowledged as the target of novel pesticide effect.Acetylglucosamine Glycosylase plays keying action in the degradation process of insect chitin, and adopt RNA perturbation technique, the application of dsRNA in pest control carrying out acetylglucosamine glycosidase genes is significant.
Summary of the invention
The present invention aims to provide the application of dsRNA and dsRNA in lethal insect of a kind of gypsymoth based on gene silent technology β-N-Acetyl-D-glucosamine glycosidase genes and lethal gene fragment wherein and this lethal gene fragment.
The present invention is achieved by the following technical solutions: gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes ldnag1, its nucleotide sequence is as shown in SEQIDNO.1.Described ldnag1 gene filters out from gypsymoth transcript profile database, and its length is 1821bp, 607 amino acid of encoding.
In addition, the invention provides a kind of gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldnag2, its nucleotide sequence is as shown in SEQIDNO.2.
Further, the invention provides above-mentioned gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe synthetic method of Nag2, the steps include: described in basis ldthe nucleotide sequence of Nag1, with sequence SEQIDNO.3 be upstream primer, sequence SEQIDNO.4 for downstream primer, will ldthe restructuring pEASY-T1 plasmid of Nag1 is as template, and pcr amplification obtains ldnag2.
Then, the present invention utilizes related kit to synthesize dsRNA further, and provides the application of this dsRNA in lethal insect: dsRNA is to gypsymoth body cavity in injection.Result shows, and: dsRNA can the mrna expression of specific reticent β-N-Acetyl-D-glucosamine glycosidase genes, gypsymoth because of difficulty of casting off a skin dead.
The present invention is according to gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes ldnag1 obtains lethal gene fragment ldnag2, and this lethal gene fragment ldthe dsRNA of Nag2, utilizes dsRNA can reach the object of Control pests, is expected to a large amount of uses reducing agricultural chemicals; protection of the environment, reduces production cost, improves economic return; there is market application foreground, for the development of the insect pest control method of safety nuisance free provides new way.
Accompanying drawing explanation
Fig. 1 be after prepupal period Lymantria dispar larvae injection dsRNA and DEPC process water, gypsymoth is grown affect comparison diagram.Result shows: the experimental group (A) of the injection dsRNA difficulty that occurs casting off a skin cannot turn to the phenotype of pupa and death, and the control group (B) of injection DEPC process water is grown normally.
Fig. 2 is for after employing real time fluorescence quantifying PCR method research prepupal period Lymantria dispar larvae injection dsRNA ldthe relative expression quantity comparison diagram of Nag2.In figure: the prepupal period larva of 1 expression control group; 2 represent injection ldthe prepupal period larva of the dsRNA of Nag2.
Embodiment
embodiment 1:gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe acquisition of Nag2
According to the gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes obtained from gypsymoth transcript profile database ldthe nucleotide sequence of Nag1, adopt the specific primer of primerpremier5.0 software design, upstream primer sequence is SEQIDNO.3, and downstream primer sequence is SEQIDNO.4, and all primers are by the synthesis of the English Weihe River, Shanghai prompt base biological company limited.By gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes that success obtains ldthe restructuring pEASY-T1 plasmid of Nag1 is as template, and pcr amplification obtains lethal gene fragment ldnag2, uses MicroEluteCyclePureKit(OMEGA) test kit carries out purifying.
embodiment 2:gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe acquisition of the dsRNA of Nag2
By gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment that embodiment 1 obtains ldnag2, according to T7RiboMAX ExpressRNAiSystem(Promega) test kit specification sheets, in-vitro transcription synthesis dsRNA.The dsRNA the obtained agarose gel electrophoresis of 1.5% detects its unicity, with microplate reader (ThermoScientificMultiskanGO, ThermoFisherScientific, USA) detect its concentration, and be concentrated into final concentration 1 μ g/ μ l be saved to-80 DEG C for subsequent use.
embodiment 3:gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe lethal gypsymoth experiment of the dsRNA of Nag2
1) gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe dsRNA injection of Nag2
Choose prepupal period larva for injecting the dsRNA of above-mentioned synthesis.The microsyringe of 5ul specification is used for injection, can not be firmly excessive during injection, and flank portion, as injection point, notes avoiding valve.The amount of injection dsRNA is 5 μ g, and arranges control group, experimental group and each 20 of the control group of the DEPC process water of injection equivalent.After injection, larva to be positioned in biochemical cultivation case with carrying out purchased from Chinese forest-science academy forest ecological environment and Protective strategy institute industry raising (illumination: interlunation=16h:8h, temperature 26 ± 1 DEG C, humidity 70%).
2) observation of gypsymoth phenotype after dsRNA is injected
Continue to raise after larva injection, and observe in time.The experimental group of injection dsRNA gypsymoth has part larva to be adult and dead because sloughing off nymphosis.
3) observation of gypsymoth death condition after dsRNA is injected
The gypsymoth experimental group mortality ratio of injection dsRNA is 25%, and this is compared with the control group (mortality ratio is 1.67%) of injection DEPC process, and lethal effect is obvious.The relative expression quantity studying β-N-Acetyl-D-glucosamine glycosidase genes in the gypsymoth experimental group prepupal period larva body showing to inject dsRNA by use real time fluorescence quantifying PCR method obviously reduces.
<110> Institutes Of Technology Of Taiyuan
<120> is based on the gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes of gene silent technology
<160>4
<170>PatentInversion3.5
<210>1
<211>1821
<212>DNA
<213> gypsymoth
<222>（1）…（1821）
<400>1
atggcgagcctatgggtgctattaactttattggctgtgaacctagtccagtccagtgaa60
aataatgtcgacaacatagtggcatctgtttatgagccatcgtggttgtacaaatgcatt120
cccgactccggttgtcagcggacatcacatccaaatccagacagctacaatgtcaccaat180
ttcatgtttaccagtatggacctgtgcagaacagtatgtgggagattcggaggtctatgg240
ccacgtccggtgtcagctgctttgagcatccaaactgttaagatacatcctaattgtctc300
aggtttgacttggaaaatgtaccagttgaagctcgggagttactggcagagatgacacaa360
atcgttggatcaaatttactggcagagtgcggtggtgaagtcacggagatggttgagaca420
cctgtcgttgtctacattaacataaaatcacctaatgtggctctaacttgggacactaat480
gaacaatatacgctcgatgtacagagtaaagagagccagatatcggtacacgtgggagct540
gatacagtgtacggagcgcgtcatgctttggagacattcacacaactgattgcatcagat600
agaccgaaatactcagatgataacaaatgcggtcttcaacttgtcgccggagctaaaatc660
cgtgatcgtccagtttataagtacagaggattattgcttgattctagtagacattttata720
ccaatgaaggatgtattgagaactatcgatggtatggccgctaataagatgaacgtcttc780
cattggcatgttactgattctcacagttttcctttggagtccactagagtaccacaattt840
acaaggtacggcgcttactctgccaaagagatctactcagtggaagaggtcaggatgttg900
ataaagtacgcacaagtgcggggtatacgtgttgtaattgaaattgatgcaccagcgcac960
gctggtaatggatggcaatggggaaaggagtatggcttcggtgatttggctgtgtgtgtg1020
aatgaatatccgtggcgtcctctttgtatagaacctccgtgcggtcagctcaacccggcg1080
aatccaacaatgtatcgtgtacttcgagatctatacagagatattgctgaaacattaccg1140
catcctgcgttactgcatatgggaggtgatgaggtgttcttcggctgctggaattccagt1200
gaagagatcacaaactatatgcaacaacaaaattacgagataaccaaagaaggatttata1260
gaattatggtctgaatttcataccaaagccctccaagcatgggacgaagagttacaagcg1320
gcaggaggtacgccgcagcccgtgatgttatggtcgtctgaactaacccaagcgcataga1380
attcagaaccatcttagcaaggataggtatgtgattcaagtatgggagcctgtaggcaat1440
ccacttctgacgcagctcatccgaatgggttaccgtgttatcagcgttccaaaagatatc1500
tggtacctggaccatggtttctggggtataactaaatactccaactggaggaagatgtac1560
gcacatacgttaccaagtgatccgaatgtattaggcggtgaagtggcaatgtggactgaa1620
tatttagatgaacaagcattggacactcgtgtatggcctcgtgcagccgcagtcgctgaa1680
agactctggtcagatccaactggtagtgtatatgccgctgaagctcgtatgcagcgtcac1740
agatctcgtcttattgcaagaggtttgcgaccggacgtcatatcaccagaatggtgctca1800
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<210>2
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<212>DNA
<213> gypsymoth
<400>2
gcacgctggtaatggatggcaatggggaaaggagtatggcttcggtgatttggctgtgtg60
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ggcgaatccaacaatgtatcgtgtacttcgagatctatacagagatattgctgaaacatt180
accgcatc188
<210>3
<211>15
<212>DNA
<213> artificial sequence
<400>3
gcacgctggtaatgg15
<210>4
<211>17
<212>DNA
<213> artificial sequence
<400>4
gatgcggtaatgtttca17

Claims (5)

1. gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes ldnag1, its nucleotide sequence is as shown in SEQIDNO.1.
2. gypsymoth β-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldnag2, its nucleotide sequence is as shown in SEQIDNO.2.
3. gypsymoth β described in claim 2-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe synthetic method of Nag2, is characterized in that, the steps include: according to claim 1 ldthe nucleotide sequence of Nag1, with sequence SEQIDNO.3 be upstream primer, sequence SEQIDNO.4 for downstream primer, will ldthe restructuring pEASY-T1 plasmid of Nag1 is as template, and pcr amplification obtains ldnag2.
4. gypsymoth β described in claim 2-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe dsRNA of Nag2.
5. gypsymoth β described in claim 4-N-Acetyl-D-glucosamine Glycosylase lethal gene fragment ldthe application of dsRNA in lethal insect of Nag2.
CN201510974582.8A 2015-12-23 2015-12-23 Gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes based on gene silent technology Active CN105400807B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061591A1 (en) * 1998-05-22 1999-12-02 Kirin Beer Kabushiki Kaisha ENDO-β-N-ACETYLGLUCOSAMINIDASE GENE
CN102586208A (en) * 2012-03-13 2012-07-18 中国农业科学院饲料研究所 Protein with Beta-N-acetamido glucosaminidase activity as well as encoding gene and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061591A1 (en) * 1998-05-22 1999-12-02 Kirin Beer Kabushiki Kaisha ENDO-β-N-ACETYLGLUCOSAMINIDASE GENE
CN102586208A (en) * 2012-03-13 2012-07-18 中国农业科学院饲料研究所 Protein with Beta-N-acetamido glucosaminidase activity as well as encoding gene and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
容烁: "飞蝗β-N-乙酰氨基葡萄糖苷酶基因的克隆、表达及功能研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
樊东 等: "八字地老虎β－Ｎ－ 乙酰葡萄糖胺糖苷酶基因cDNA序列的克隆及原核表达研究", 《植物保护》 *
贺玉年 等: "昆虫β-N-乙酰葡萄糖胺糖苷酶研究进展", 《东北农业大学学报》 *

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