CN105238786B - Striped rice borer lethal gene csu-miR-9b and its application - Google Patents
Striped rice borer lethal gene csu-miR-9b and its application Download PDFInfo
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- CN105238786B CN105238786B CN201510674017.XA CN201510674017A CN105238786B CN 105238786 B CN105238786 B CN 105238786B CN 201510674017 A CN201510674017 A CN 201510674017A CN 105238786 B CN105238786 B CN 105238786B
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- mir
- csu
- rice borer
- striped rice
- gene
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- 241000426497 Chilo suppressalis Species 0.000 title claims abstract description 31
- 108700005090 Lethal Genes Proteins 0.000 title abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 4
- 239000002679 microRNA Substances 0.000 abstract description 16
- 108091070501 miRNA Proteins 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 3
- 108091027559 Mir-96 microRNA Proteins 0.000 abstract description 3
- 230000001665 lethal effect Effects 0.000 abstract description 3
- 108091086713 miR-96 stem-loop Proteins 0.000 abstract description 3
- 108091070961 miR-96-3 stem-loop Proteins 0.000 abstract description 3
- 238000007622 bioinformatic analysis Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 108091041015 miR-9b-1 stem-loop Proteins 0.000 abstract 5
- 108091051156 miR-9b-2 stem-loop Proteins 0.000 abstract 5
- 108091060513 miR-9b-4 stem-loop Proteins 0.000 abstract 5
- 108091058949 miR-9b-5 stem-loop Proteins 0.000 abstract 5
- 230000000694 effects Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000426499 Chilo Species 0.000 description 3
- 108700011259 MicroRNAs Proteins 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 239000000575 pesticide Substances 0.000 description 3
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 241000255777 Lepidoptera Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091030146 MiRBase Proteins 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
- 238000012271 agricultural production Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015556 catabolic process 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
- 238000006731 degradation reaction Methods 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
- 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
- 230000003252 repetitive 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
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
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Abstract
The invention discloses striped rice borer lethal gene csu miR 9b and its applications.Striped rice borer lethal gene csu miR 9b, 5 ' UCUUUGGUUAUCUAGCUGUAUGA 3 ' of sequence.Applications of the csu miR 9b of the present invention in striped rice borer deinsectization reagent is prepared.The present invention utilizes complete miRNA bioinformatic analysis, to search an effective miRNA from the tiny RNA literature data of magnanimity --- csu miR 9b.Striped rice borer is injected using the csu miR 9b synthesized in vitro, there is apparent lethal effect to striped rice borer, illustrate that the miR-96 gene can be as effective chaff interferent of striped rice borer important gene.
Description
Technical field
The invention belongs to agricultural biological technical fields, are related to striped rice borer lethal gene csu-miR-9b and its application.
Background technology
Striped rice borer Chilo suppressalis (Walker) belong to Lepidoptera, and Pyralidae is endangered the most on China's rice
One of serious Occurrence.In China, the continuous single long-time service of chemical agent has caused striped rice borer to produce Multiple Pesticides
Different degrees of resistance has been given birth to, has needed to continue to increase Pesticide use amount and can be only achieved satisfied control effect, has been caused more tight
The environmental pollution of weight forms vicious circle.Therefore, in agricultural production practice, it is badly in need of the replacement except chemical pesticide and prevents hand
Section.
MicroRNAs (miRNAs) is a kind of small, similar to the molecule of siRNA, is compiled by higher eucaryote genome
Code, miRNA mRNA or hinder its translation by silencing complex (RISC) being guided to degrade with target gene mRNA base pairings, so as to
In growth, development, the metabolism of post-transcriptional level regulation and control biology.MiRNAs is quite conservative in spore, plant, animal and
Only in specific tissue and stage of development expression, the tissue specificity and timing of miRNA determine the miRNAs found in fungi
The specific Function of tissue and cell, shows that miRNA plays a variety of adjustment effects in cell growth and growth course.
In organism, it is necessary that some important gene pairs, which sustain life,.Therefore, for theoretically, if sharp
The expression of important gene in agricultural pests is interfered with miRNA analogs, then can cause the teratogenesis or lethal of pest, so as to
Achieve the purpose that control pest.
Invention content
The purpose of the present invention is being directed to the above-mentioned deficiency of the prior art, striped rice borer lethal gene csu-miR-9b is provided.
It is a further object of the present invention to provide the applications of striped rice borer lethal gene csu-miR-9b.
The purpose of the present invention can be achieved through the following technical solutions：
Striped rice borer lethal gene csu-miR-9b, sequence 5 '-UCUUUGGUUAUCUAGCUGUAUGA-3 ' (SEQ ID
NO.1)。
The Csu-miR-9b is had found by following bioinformatics method analysis：
(1) contaminated sequence and nonsense sequence are removed, the shorter than sequence of 18nt is filtered out, obtains the clean number that can be marked
According to；
(2) using Genbank (http://www.ncbi.nlm.nih.gov/genbank/) sequence alignment is carried out, it uses
Rfam(http://rfam.sanger.ac.uk) the just extron of removal, antisense extron, just introne, antisense include
Son, rRNA, tRNA etc..Repetitive sequence is removed using RepBase (www.girinst.org/repbase)；
(3) in known striped rice borer genome (http://ento.njau.edu.cn/ChiloDB/) in the case of, it uses
MiRDeep predicts miRNA.First use MegaBlast (http://www.mega-blast.com/) it will treated tiny RNA library
Sequence is mapped on genome, reuses miRDeep, and miRNA predictions are carried out with default parameters；
(4) in order to analyze the family of miRNA and conservative, we use Patscan, with 211 parameters that striped rice borer is ripe
Body miRNA is compared with the known miRNA on miRBase version 19.0, finally obtains Csu-miR-9b.
Applications of the csu-miR-9b of the present invention in striped rice borer deinsectization reagent is prepared.
A kind of striped rice borer deinsectization method, with the solution of the csu-miR-9b in the upper back of Chilo spp larvae or side
It is injected or using the csu-miR-9b feeding striped rice borer.
The deinsectization method, preferably comprises following steps：
(1) csu-miR-9b to a concentration of 200pmol/ μ L described in claim 1 is diluted with nuclease-free water；
(2) Chilo spp larvae of suitable number of days is chosen, in upper back or side, 0.5 μ L/ heads are injected using micro syringe
Csu-miR-9b solution；
(3) polypide is provoked with syringe needle, waited for a period of time, treated that csu-miR-9b has been mixed into larva haemocoele, gently extract needle
Head.
Advantageous effect：
The present invention utilizes complete miRNA bioinformatic analysis, and one is searched from the tiny RNA literature data of magnanimity
Effective miRNA --- csu-miR-9b.Striped rice borer is injected using the csu-miR-9b synthesized in vitro, is had to striped rice borer apparent
Lethal effect illustrates that the miR-96 gene can be as effective chaff interferent of striped rice borer important gene.
The csu-miR-9b that the present invention synthesizes can effectively inhibit the expression of important gene in striped rice borer body, better against
The degradation of RNA enzyme, simultaneously synthesizing cost is relatively low, is used convenient for many experiments.
Description of the drawings
Fig. 1 csu-miR-9b injection experiment lethality statistical charts.
Specific embodiment
Embodiment 1
(1) chemical synthesis striped rice borer lethal gene csu-miR-9b, 5 '-UCUUUGGUUAUCUAGCUGUAUGA- of sequence
3’；
(2) it is purified using high performance liquid chromatography (HPLC)；
(3) using spectrophotometer accurate quantification, freeze-drying preserves.
Embodiment 2
A kind of lethal gene csu-miR-9b injecting methods of striped rice borer, comprise the following steps：
(1) csu-miR-9b to a concentration of 200pmol/ μ L described in claim 1 is diluted with nuclease-free water；
(2) Chilo spp larvae of suitable number of days is chosen, in upper back or side, 0.5 μ L/ heads are injected using micro syringe
Csu-miR-9b solution；
(3) polypide is provoked with syringe needle, waited for a period of time, treated that csu-miR-9b has been mixed into larva haemocoele, gently extract needle
Head；
(4) larva after injection is placed in 12 orifice plates, adds in appropriate rice seedlings, close the lid, tied with rubber band,
Phenotype was observed every 6 hours, the results are shown in Figure 1, it is seen that csu-miR-9b injects striped rice borer, has apparent cause to striped rice borer
Dead effect illustrates that the miR-96 gene can be as effective chaff interferent of striped rice borer important gene.
Claims (2)
- Applications of the 1.csu-miR-9b in striped rice borer deinsectization reagent is prepared；The csu-miR-9b sequences for 5 '- UCUUUGGUUAUCUAGCUGUAUGA -3’。
- 2. a kind of striped rice borer deinsectization method, it is characterised in that use csu-miR-9b feeding striped rice borer, the csu-miR-9b Sequence is 5 '-UCUUUGGUUAUCUAGCUGUAUGA -3 '.
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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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| CN105238786A CN105238786A (en) | 2016-01-13 |
| CN105238786B true CN105238786B (en) | 2018-06-29 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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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| 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 |
|---|
| ChiloDB:a genomic and transcriptome database for an important rice insect pest Chilo suppressalis;Yin C. et al;《Database(Oxford)》;20140704;第2014卷;第1-7页 * |
| Lagos-Quintana M et al.;MIMAT0000114;《miRBase》;20011231;第1页 * |
| MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila;Yan Li et al.;《Genes & Development》;20061002;第20卷(第20期);摘要，第2802页倒数第1段，图1 * |
| 五种农业害虫微小RNA基因的生物信息学预测与分析;余论;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140415;第2014卷(第04期);D046-12 * |
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