CN109385422A - A kind of pair of striped rice borer be lethal and insect specificity Csu-miR-14 gene and its application of delayed development - Google Patents

A kind of pair of striped rice borer be lethal and insect specificity Csu-miR-14 gene and its application of delayed development Download PDF

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Publication number
CN109385422A
CN109385422A CN201710674376.4A CN201710674376A CN109385422A CN 109385422 A CN109385422 A CN 109385422A CN 201710674376 A CN201710674376 A CN 201710674376A CN 109385422 A CN109385422 A CN 109385422A
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gene
mir
csu
lethal
rice borer
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李飞
肖花美
贺康
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Zhejiang University ZJU
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Zhejiang University ZJU
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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
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
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  • Molecular Biology (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Present invention discloses a kind of pair of striped rice borer be lethal and the insect specificity of delayed developmentCsu‑miR‑14Gene and its application, pass through byCsu‑miR‑14Gene, which is transferred in wild rice, to be spent in 11 kinds, by the continuous feeding Chilo spp larvae of the transgenic paddy rice, achievees the effect that lethal striped rice borer and delayed development.

Description

A kind of pair of striped rice borer be lethal and the insect specificity Csu-miR-14 gene of delayed development And its application
Technical field
The invention belongs to agricultural biological technical field, it is specifically related to the insectean metamorphosis development regulated and controled based on post-transcriptional level MiR-96 gene Csu-miR-14 and its sequence.
Background technique
Striped rice borer Chilo suppressalis (Walker) belongs to Lepidoptera, and Pyralidae is endangered the most on China's rice One of serious Occurrence.In China, continuous single long-term a large amount of use of chemical pesticide has caused striped rice borer to a variety of agricultures Medicine produces different degrees of resistance, and needing to continue to increase Pesticide use amount can be only achieved satisfied control efficiency, causes more For serious environmental pollution, vicious circle is formed.Therefore, in agricultural production practice, the substitution being badly in need of except chemical pesticide is anti- Control means.
MicroRNAs (miRNAs) is a kind of small RNA molecular that endogenous length is 22-25nt, by higher eucaryote Genome encoding leads to target gene silencing by mRNA shearing or the translation negative regulation target gene of suppression target protein, from And the weight such as development, cell differentiation, proliferation and the apoptosis of body, lipid metabolism, hormone secretion is participated in and regulated and controled in post-transcriptional level Want bioprocess.The change of the development by metamorphosis of insect periodically husking, body size and form by moulting hormone (Ecdysone, E it) is adjusted jointly with juvenile hormone (Juvenile hormones, JH).It can be simultaneously research shows that there are part miRNA in insect Multiple genes of Targeted-control moulting hormone signal path, such as miR-14, and then influence the generation of insect molting and metamorphosis.
Therefore, the specific miRNA for participating in regulation agricultural pests development by metamorphosis important gene is existed using genetically modified plants It is overexpressed in plant, teratogenesis, lethal or development slow can be then caused after pests, to reach the mesh of control pest 's.
The present invention by existing insect miR-96 gene family's family sequence analyze, obtain insect specificity miRNA, using turn Csu-miR-14 gene is transferred in wild rice and spends in 11 kinds by the method for gene, by the continuous feeding two of the transgenic paddy rice Change snout moth's larva larva, achievees the effect that lethal striped rice borer and delayed development.
Summary of the invention
The object of the present invention is to provide a kind of pair of striped rice borer be lethal and the insect specificity Csu-miR-14 base of delayed development Cause reaches the lethal and delayed development effect to striped rice borer using Csu-miR-14 trans-genetic hybrid rice feeding striped rice borer is turned.
The present invention is directed to obtain the miRNA target gene that can be used in agricultural production controlling rice grub.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of pair of striped rice borer be lethal and the insect specificity Csu-miR-14 gene of delayed development, which is characterized in that described Gene order are as follows: 5 '-TCAGTCTTTTTTCTCTCTCCTAT-3 '；
Csu-miR-14 gene of the present invention is in lethal striped rice borer and/or delays the developmental application of striped rice borer.
Beneficial effects of the present invention:
1. Csu-miR-14 gene of the invention is transferred in rice, then with transgenic paddy rice feeding striped rice borer, discovery Have the effect of that apparent lethal and delayed development substantially to no effect to brown paddy plant hopper illustrates Csu- of the invention to striped rice borer MiR-14 gene can be used as the specific target gene that control striped rice borer is caused harm.
Specific embodiment
Embodiment 1
The experiment of transgenic paddy rice feeding:
(1) Csu-miR-14 gene of the invention is transferred in wild rice and is spent in 11, chosen tillering stage 90 days 12 plants of transgenic paddy rices are cut rice strain with scissors by spending 11 in PCR test positive and 6 plants of wild rices as control It to 4cm length, is placed in 9cm culture dish, each culture dish is put into 6-8 intercept；
(2) newly hatched larvae hatched striped rice borer with fine, soft fur pen in for 24 hours is connected to rice stem intercept, each culture dish access 20 larvas splitted rice stem intercept every 3 days, and test worm is transferred to fresh rice stem and is truncated, guarantees that striped rice borer has Enough fresh foods observe and record the striped rice borer husking parameters such as developmental condition and the death rate until pupating；
(3) parallel control is set: Csu-miR-14 gene is substituted for Sex-miR-14 gene 5 '-respectively TCAGTCTTTTTCTCTCTCCTA-3 ', Csu-novel-miR15 gene 5 '-TATAACTATAGATAGTACAGT-3 ', then Repeat the above steps (1), (2) respectively, obtains control 1, control 2.
The striped rice borer death rate and the average change into pupa rate after the different transgenic paddy rice feedings of table 1
As can be seen from the data in table 1, Csu-miR-14 gene of the invention be transferred to after rice to the lethal effect of striped rice borer, Delayed development significant effect.
Embodiment 2
(1) Csu-miR-14 gene of the invention is transferred in wild rice and is spent in 11, chosen tillering stage 90 days 12 plants of transgenic paddy rices are by spending 11 6 plants as control in PCR test positive and wild rice, even root takes rice straw containing 2- 3 plants, partial blade is cut off, is placed in 20*2.5cm Boiling tube, and sewed it up with gauze；
(2) it takes 3 age brown paddy plant hopper nymphs to be carefully connected to rice stem with pest sucking device, accesses 15 nymphs in each test tube, if 3 repetitions are set, the development of statistics brown paddy plant hopper and death condition is observed every 12h, until all sprouting wings, during which rice is kept to have enough Moisture.
The brown paddy plant hopper death rate and the average change into pupa rate after 2 transgenic paddy rice feeding of table
By the data of table 2 it is found that after Csu-miR-14 gene of the invention is transferred to rice, brown paddy plant hopper feeding transgenosis water The death rate, the average change into pupa rate and the basic indifference of feeding non-transgenic rice after rice, it is seen that Csu-miR-14 gene of the invention Substantially to no effect to brown paddy plant hopper, Csu-miR-14 gene specific is stronger, to striped rice borer excellent effect.

Claims (2)

1. a kind of pair of striped rice borer be lethal and the insect specificity of delayed developmentCsu-miR-14Gene, which is characterized in that the base Because of sequence are as follows: 5 '-TCAGTCTTTTTTCTCTCTCCTAT-3 '.
2. as described in claim 1Csu-miR-14Gene is in lethal striped rice borer and/or delays the developmental application of striped rice borer.
CN201710674376.4A 2017-08-09 2017-08-09 A kind of pair of striped rice borer be lethal and insect specificity Csu-miR-14 gene and its application of delayed development Pending CN109385422A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690651A (en) * 2020-07-02 2020-09-22 华中农业大学 Truncated miRNA for blocking ecdysone synthesis pathway of chilo suppressalis in rice and application thereof

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US20080026382A1 (en) * 2006-07-31 2008-01-31 Hui Wang Enzymatic labeling of RNA
CN104372007A (en) * 2014-11-27 2015-02-25 深圳市第二人民医院 Osteosarcoma biomarker and application thereof
CN104673827A (en) * 2013-12-02 2015-06-03 华中农业大学 Application of Chilo suppressalis endogenesis small RNA in rice inset resistance improvement
CN105165885A (en) * 2015-09-16 2015-12-23 华南师范大学 Application of miR-375-3p in prevention and treatment of lepidoptera pests
CN105238786A (en) * 2015-10-16 2016-01-13 南京农业大学 Chilo suppressalis lethal gene csu-miR-9b and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080026382A1 (en) * 2006-07-31 2008-01-31 Hui Wang Enzymatic labeling of RNA
CN104673827A (en) * 2013-12-02 2015-06-03 华中农业大学 Application of Chilo suppressalis endogenesis small RNA in rice inset resistance improvement
CN104372007A (en) * 2014-11-27 2015-02-25 深圳市第二人民医院 Osteosarcoma biomarker and application thereof
CN105165885A (en) * 2015-09-16 2015-12-23 华南师范大学 Application of miR-375-3p in prevention and treatment of lepidoptera pests
CN105238786A (en) * 2015-10-16 2016-01-13 南京农业大学 Chilo suppressalis lethal gene csu-miR-9b and application thereof

Non-Patent Citations (3)

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KANG HE等: ""Transgenic microRNA-14 rice shows high resistance to rice stem borer"", 《PLANT BIOTECHNOLOGY JOURNAL》 *
孙杨: ""二化螟蛋白编码基因和miRNA在化蛹和蛹期发育中的功能分析"", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 *
肖花美: ""五种昆虫非编码RNA基因发现及miR-14的功能研究"", 《中国博士学位论文全文数据库（电子期刊）基础科学辑》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111690651A (en) * 2020-07-02 2020-09-22 华中农业大学 Truncated miRNA for blocking ecdysone synthesis pathway of chilo suppressalis in rice and application thereof

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Application publication date: 20190226