CN105238786A - Chilo suppressalis lethal gene csu-miR-9b and application thereof - Google Patents
Chilo suppressalis lethal gene csu-miR-9b and application thereof Download PDFInfo
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- CN105238786A CN105238786A CN201510674017.XA CN201510674017A CN105238786A CN 105238786 A CN105238786 A CN 105238786A CN 201510674017 A CN201510674017 A CN 201510674017A CN 105238786 A CN105238786 A CN 105238786A
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- Prior art keywords
- mir
- csu
- chilo suppressalis
- rice borer
- striped rice
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- 241000426497 Chilo suppressalis Species 0.000 title claims abstract description 32
- 108700005090 Lethal Genes Proteins 0.000 title claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 6
- 241000426499 Chilo Species 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 101710163270 Nuclease Proteins 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000002679 microRNA Substances 0.000 abstract description 17
- 108091070501 miRNA Proteins 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 230000001665 lethal effect Effects 0.000 abstract description 4
- 238000003766 bioinformatics method Methods 0.000 abstract description 2
- 108091032955 Bacterial small RNA Proteins 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 230000000749 insecticidal effect Effects 0.000 abstract 1
- 108700011259 MicroRNAs Proteins 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108091027559 Mir-96 microRNA Proteins 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 108091086713 miR-96 stem-loop Proteins 0.000 description 2
- 108091070961 miR-96-3 stem-loop Proteins 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000255893 Pyralidae Species 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a chilo suppressalis lethal gene csu-miR-9b and application thereof. The sequence of the chilo suppressalis lethal gene csu-miR-9b is shown as 5'-UCUUUGGUUAUCUAGCUGUAUGA-3'. The invention discloses application of the csu-miR-9b in preparing a chilo suppressalis insecticidal reagent. On the basis of complete miRNA bioinformatics analysis, an effective miRNA, namely csu-miR-9b, is searched from mass data in a small RNA library. The csu-miR-9b synthesized in vitro, injected to chilo suppressalis, has a significant lethal effect on the chilo suppressalis, showing that the miRNA can serve as an effective interferent for important genes of the chilo suppressalis.
Description
Technical field
The invention belongs to agricultural biological technical field, relate to striped rice borer lethal gene csu-miR-9b and application thereof.
Background technology
Striped rice borer Chilosuppressalis (Walker) belongs to lepidopteran, Pyralidae, is China paddy rice endangers one of Occurrence the most serious.In China, the continuous single life-time service of chemical agent has caused striped rice borer 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.Therefore, in agricultural production practice, be badly in need of the alternative means of prevention outside chemical pesticide.
MicroRNAs (miRNAs) is a kind of little, be similar to the molecule of siRNA, by higher eucaryote genome encoding, miRNA by and target gene mRNA base pairing guide silencing complex (RISC) to degrade mRNA or hinder it translate, thus in the growth of post-transcriptional level regulation and control biology, growth, metabolism.MiRNAs is quite conservative in spore, the miRNAs found in plant, animal and fungi only expresses at specific tissue and etap, the tissue specificity of miRNA and timing, determine the specific Function of tissue and cell, show that miRNA plays multiple regulating effect in Growth of Cells and growth course.
In organism, it is necessary that some important gene pairss sustain life.Therefore, theoretically, if utilize miRNA analogue 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.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, striped rice borer lethal gene csu-miR-9b is provided.
Another object of the present invention is to provide the application of this striped rice borer lethal gene csu-miR-9b.
Object of the present invention realizes by following technical scheme:
Striped rice borer lethal gene csu-miR-9b, sequence is 5 '-UCUUUGGUUAUCUAGCUGUAUGA-3 ' (SEQIDNO.1).
Described Csu-miR-9b is found by following bioinformatics method analysis:
(1) remove contaminated sequence and nonsense sequence, filter out the sequence being shorter than 18nt, obtain the clean data that can mark;
(2) use Genbank (http://www.ncbi.nlm.nih.gov/genbank/) to carry out sequence alignment, use Rfam (http://rfam.sanger.ac.uk) to remove just exon, antisense exon, just intron, antisense intron, rRNA, tRNA etc.RepBase (www.girinst.org/repbase) is used to remove tumor-necrosis factor glycoproteins;
(3) when known striped rice borer genome (http://ento.njau.edu.cn/ChiloDB/), miRDeep is used to predict miRNA.First use MegaBlast (http://www.mega-blast.com/) to be mapped on genome by the tiny RNA library sequence after process, re-use miRDeep, carry out miRNA prediction with default parameters;
(4) in order to analyze family and the conservative property of miRNA, we use Patscan, compared by the known miRNA on ripe for striped rice borer body miRNA and miRBaseversion19.0, finally obtain Csu-miR-9b with 211 parameters.
The application of csu-miR-9b of the present invention in preparation striped rice borer deinsectization reagent.
A kind of striped rice borer deinsectization method, carries out injecting or use described csu-miR-9b feeding striped rice borer at the upper back of Chilo spp larvae or side with the solution of described csu-miR-9b.
Described deinsectization method, preferably comprises following steps:
(1) diluting csu-miR-9b according to claim 1 to concentration with nuclease free water is 200pmol/ μ L;
(2) choose the Chilo spp larvae of suitable number of days, in upper back or side, use microsyringe to inject 0.5 μ L/ head csu-miR-9b solution;
(3) provoke polypide with syringe needle, wait for a period of time, treat that csu-miR-9b has been mixed into larva haemocoele, extract syringe needle gently.
Beneficial effect:
The present invention utilizes complete miRNA bioinformatic analysis, searches an effective miRNA from the tiny RNA literature data of magnanimity---csu-miR-9b.Utilize the csu-miR-9b of external synthesis to inject striped rice borer, to striped rice borer, there is obvious lethal effect, illustrate that this miR-96 gene can be used as effective chaff interference of striped rice borer important gene.
The csu-miR-9b of the present invention's synthesis effectively can suppress the expression of important gene in striped rice borer body, resists the degraded of RNA enzyme better, synthesizes cost lower simultaneously, is convenient to great many of experiments and uses.
Accompanying drawing explanation
Fig. 1 csu-miR-9b injects experiment lethality rate statistical graph.
Embodiment
Embodiment 1
(1) chemosynthesis striped rice borer lethal gene csu-miR-9b, sequence is 5 '-UCUUUGGUUAUCUAGCUGUAUGA-3 ';
(2) high performance liquid chromatography (HPLC) is used to carry out purifying;
(3) use spectrophotometer accurate quantification, freeze-drying is preserved.
Embodiment 2
A lethal gene csu-miR-9b injecting method for striped rice borer, comprises following steps:
(1) diluting csu-miR-9b according to claim 1 to concentration with nuclease free water is 200pmol/ μ L;
(2) choose the Chilo spp larvae of suitable number of days, in upper back or side, use microsyringe to inject 0.5 μ L/ head csu-miR-9b solution;
(3) provoke polypide with syringe needle, wait for a period of time, treat that csu-miR-9b has been mixed into larva haemocoele, extract syringe needle gently;
(4) larva after injection is placed in 12 orifice plates, add suitable quantity of water rice seedling, cover lid, tie with bungee, observed phenotype every 6 hours, as shown in Figure 1, visible csu-miR-9b injects striped rice borer to result, to striped rice borer, there is obvious lethal effect, illustrate that this miR-96 gene can be used as effective chaff interference of striped rice borer important gene.
Claims (4)
1. striped rice borer lethal gene csu-miR-9b, is characterized in that sequence is 5 '-UCUUUGGUUAUCUAGCUGUAUGA-3 '.
2. the application of csu-miR-9b according to claim 1 in preparation striped rice borer deinsectization reagent.
3. a striped rice borer deinsectization method, is characterized in that carrying out with the solution of csu-miR-9b according to claim 1 the csu-miR-9b feeding striped rice borer injected or use described in claim 1 at the upper back of Chilo spp larvae or side.
4. deinsectization method according to claim 3, is characterized in that comprising following steps:
(1) diluting csu-miR-9b according to claim 1 to concentration with nuclease free water is 200pmol/ μ L;
(2) choose the Chilo spp larvae of suitable number of days, in upper back or side, use microsyringe to inject 0.5 μ L/ head csu-miR-9b solution;
(3) provoke polypide with syringe needle, wait for a period of time, treat that csu-miR-9b has been mixed into larva haemocoele, extract syringe needle gently.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510674017.XA CN105238786B (en) | 2015-10-16 | 2015-10-16 | Striped rice borer lethal gene csu-miR-9b and its application |
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| CN201510674017.XA CN105238786B (en) | 2015-10-16 | 2015-10-16 | Striped rice borer lethal gene csu-miR-9b and its application |
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| Publication Number | Publication Date |
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| CN105238786A true CN105238786A (en) | 2016-01-13 |
| CN105238786B CN105238786B (en) | 2018-06-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913696A (en) * | 2018-07-30 | 2018-11-30 | 杭州贝英福生物科技有限公司 | A kind of miRNA of lethal striped rice borer and its application |
| CN109385422A (en) * | 2017-08-09 | 2019-02-26 | 浙江大学 | A kind of pair of striped rice borer be lethal and insect specificity Csu-miR-14 gene and its application of delayed development |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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2015
- 2015-10-16 CN CN201510674017.XA patent/CN105238786B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| MIMAT0000114: "Lagos-Quintana M et al.", 《MIRBASE》 * |
| YAN LI ET AL.: "MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila", 《GENES & DEVELOPMENT》 * |
| YIN C. ET AL: "ChiloDB:a genomic and transcriptome database for an important rice insect pest Chilo suppressalis", 《DATABASE(OXFORD)》 * |
| 余论: "五种农业害虫微小RNA基因的生物信息学预测与分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109385422A (en) * | 2017-08-09 | 2019-02-26 | 浙江大学 | A kind of pair of striped rice borer be lethal and insect specificity Csu-miR-14 gene and its application of delayed development |
| CN108913696A (en) * | 2018-07-30 | 2018-11-30 | 杭州贝英福生物科技有限公司 | A kind of miRNA of lethal striped rice borer and its application |
| CN108913696B (en) * | 2018-07-30 | 2020-07-03 | 杭州贝英福生物科技有限公司 | MiRNA (micro ribonucleic acid) capable of killing chilo suppressalis and application of miRNA |
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| Publication number | Publication date |
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| CN105238786B (en) | 2018-06-29 |
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