CN104673827B - Application of the endogenous tiny RNA of striped rice borer in the pest-resistant improvement of paddy rice - Google Patents
Application of the endogenous tiny RNA of striped rice borer in the pest-resistant improvement of paddy rice Download PDFInfo
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Abstract
The invention belongs to field of plant genetic.Specifically related to striped rice borer [Chilo suppressalis（Walker）Application of the endogenous tiny RNA in the pest-resistant improvement of paddy rice.The present invention is to the striped rice borer that is obtained] endogenous tiny RNA sequence carried out functional verification and application study.The means with bioinformatic analysis are sequenced using high flux tiny RNA, the endogenous tiny RNA csu 15 of striped rice borer DNA sequence dna, its nucleotide sequence such as SEQ ID NO is obtained：Shown in 1.Utilize the artificial microRNA of csu 15 sequence construct（amiRNA）Expression vector and rice transformation.In vitro stalk connects worm experiment and shows that transgenic paddy rice can significantly inhibit the growth of Chilo spp larvae.
Description
Technical field
The invention belongs to field of plant genetic.Specifically related to the endogenous tiny RNA of striped rice borer is in the pest-resistant improvement of paddy rice
In application.Clone obtains a striped rice borer [Chilo suppressalis（Walker）] endogenous tiny RNA sequence, to the sequence
Functional verification and the application study in the pest-resistant improvement of paddy rice.
Background technology
Paddy rice is one of staple food crop in the world, there are about the world population of half using it as staple food.In agricultural production
On, pest and disease damage can cause the extensive underproduction of Rice Production, cause huge economic loss.Striped rice borer is the primary pest of paddy rice
One of, it is distributed widely in the countries such as Asia, Oceania, north African, southern Europe；In China in addition to Qinghai and Tibet, there are two changes each province
The generation of snout moth's larva（Sheng Chengfa etc. is 2003).In recent years, the harm of striped rice borer is in rising trend in the most rice regions of China（Chen Hao etc.
2010）.In Rice Production, chemical reagent is the Main Means of striped rice borer preventing and treating, but people are also more passed through trying to explore other
The method that Ji and low environment endanger.Application of the Bt genes on paddy rice has preferable preventing and treating, but its for the striped rice borer of Lepidoptera
He is also constantly being attempted in case striped rice borer produces resistance in the screening pressure of Bt paddy rice new method.
RNA interference phenomenon（RNA interference,RNAi）Most early in being found in morning glory（Napoli et
al.1990）, and by Fire et al. clearly by its entitled RNAi（Fire et al.1998）.After RNAi mechanism is found, extensively
It is used in by general in gene therapy, functional genome research and breed improvement.In recent years, people are attempted with RNAi technology in plant
Applied in terms of breeding for pest resistance, transgenic arabidopsis and tobacco feeding bollworm of the Mao et al. with expression dsRNA find it
Plant pair bollworm is resistant（Mao et al.2007）；The dsRNA feeding western corn roots that Baum et al. is manually synthesized
Worm（western corn rootworm）, after 12h to 1d, the target gene expression of insect is remarkably decreased（Baum et
al.2007）.
MiRNA is the non-coding that a class is about 21~28nt（non-coding）RNA（Lagos-Quintana et
al.2001;Lau et al.2001;Lee and Ambros2001;Couzin2002), before 60~80nt hair clip
Body pre-miRNA.General pre-miRNA length is more constant in animal, but the variability of its length is larger in plant,
Can be from tens to hundreds of nt（Reinhart et al.2002;LAGOS-QUINTANA et al.2003).MiRNA's is main
Function is to play a part of gene expression regulation in biology growing and growth course, is such as led in the miRNA lin-4 of nematode
Cross the non-translational region incomplete pairing with target mRNA 3 ' ends after translating target mRNA to suppress, lin-4's is prominent
Become the change for often resulting in elegans development form（Banerjee and Slack2002;Pasquinelli and
Ruvkun2002）New miRNA discovery can be obtained by Direct Cloning or bioinformatic analysis.The wherein method of Direct Cloning
Take time and effort, people are more likely to find new miRNA by the method for bioinformatics.The method of bioinformatics need according to
By high throughput sequencing technologies, it once can carry out sequencing to the DNA molecular of hundreds of thousands to millions of.Using in synthesis
Sequencing（sequencing by synthesis）Strategy can reduce the region missing caused by secondary structure, and with required
Sample size is few, high flux, high precision, the features such as possessing automation platform simple to operation and be powerful.At present, much
Species are found that many new miRNA by the method for high-flux sequence and are uploaded to miRNA databases miRBase
（Kozomara and Griffiths-Jones2011）, such as by tiny RNA high-flux sequence, 55 are found in locust
MiRNA sequence（Wen et al.2009）, 37 new miRNA are determined in pine wood nematode（Huang et al.2010）,
777 new miRNA are found that in 10 samples in hog（Li et al.2010）Deng.
By the use of natural Mirnas of plant precursor as skeleton, the 21nt for target gene of engineer sequence
Corresponding natural miRNA sequence is replaced, the artificial mi RNA of maturation can be generated by following natural miRNA biology Forming Mechanism
（artificial microRNA,amiRNA）.Then amiRNA can as natural miRNA specifically silence target base
The expression of cause.At present, it has been applied successfully in the various plants such as arabidopsis, tobacco, tomato, paddy rice, liver moss and algae
AmiRNA technologies specifically suppress endogenous or foreign gene expression（Niu et al.2006;Schwab et al.2006;
Qu et al.2007;Khraiwesh et al.2008;Warthmann et al.2008;Molnar et al.2009）.
AmiRNA interference has higher specificity compared with dsRNA interferes, and is difficult to cause the phenomenon of " missing the target ", is described as " second generation "
Gene silent technology（Tang et al.2007）.The current technology is widely used in functional genome research, breed improvement and doctor
Treatment field, but amiRNA Technology applications have not been reported in transgenic pest-resistant breeding.The present invention uses amiRNA technologies, and two are changed
Small RNA fragments overexpression in transgenic paddy rice in source in snout moth's larva body.By striped rice borer feeding test, it is found that the transgenic paddy rice has
There is certain anti-striped rice borer effect, the research provides new approaches for plant transgene breeding for pest resistance system, also to understand in depth
The mechanism of miRNA interference provides help.
The content of the invention
It is an object of the invention to overcome the defect of prior art, identified using high throughput sequencing technologies and bioinformatics
Go out the endogenous special tiny RNA csu-15 of striped rice borer, using the corresponding amiRNA expression vectors of csu-15 sequence external structure,
And be transformed into rice varieties and spent in 11 by agrobcterium-mediated transformation, spent in transgenosis 11 overexpressions with
The consistent amiRNA of csu-15 sequences, striped rice borer is taken food after transgenic paddy rice, takes in substantial amounts of amiRNA, due to amiRNA with it is interior
Source tiny RNA csu-15 sequence is consistent, the disorder that the internal corresponding gene of striped rice borer may be caused to regulate and control, and reaches two changes of influence
Snout moth's larva grows, so as to play pest-resistant effect.
Technical scheme is as follows：
The sample in period 1. collection 6 differences of striped rice borer are grown, including ovum, 1-2 instar larvaes, 3-4 instar larvaes, 5 ages
Larva, pupa and adult, the total serum IgE of 6 samples is extracted with Trizol reagents respectively（See embodiment 1）.
2. sending Huada gene company to carry out tiny RNA high-flux sequence the total serum IgE for 6 samples of striped rice borer being collected into, lead to
Cross bioinformatics method analysis, predict in 6 samples guard miRNA, rRNA, scRNA, snoRNA, snRNA, tRNA,
The tiny RNAs such as piRNA, while undiscovered new miRNA in 6 samples is analyzed, and its expression in 6 samples（See
Embodiment 2）.Wherein csu-15 is the new miRNA of a prediction, and it exists in the midgut tissue of striped rice borer, its sequence such as sequence
List SEQ ID NO：Shown in 1, its natural precursor sequence such as sequence table SEQ ID NO：Shown in 2.
3. couple csu-15 predicted, it RT-PCR products are sequenced by stem ring RT-PCR and again can verify csu-
15 are really present in striped rice borer body（See embodiment 3）.
4. according to Warthmann etc.（2008）Report, according to the corresponding amiRNA tables of csu-15 sequence external structure
Up to carrier（See embodiment 4）, and spend 11 with Agrobacterium-medialed transformation method Introduced into Rice kind（See embodiment 5）, obtain
Transgenic rice plant.
5. pair transgenic rice plant is carried out after positive detection and the detection of amiRNA expression quantity（See embodiment 6）, choose
The high plant of amiRNA expression quantity carries out striped rice borer and connects worm identification（See embodiment 7）, obtain having obvious suppression to striped rice borer growth
The transfer-gen plant of effect.
Concrete technical scheme of the present invention is as follows：
A kind of tiny RNA csu-15 genes for suppressing the growth of rice grub striped rice borer are in transgenic pest-resistant rice is cultivated
Using the nucleotide sequence such as SEQ NO of the gene：Shown in 1.
The specific steps of application described in said gene include：
Amplimer 15-I, 15-II, 15-III, 15-IV, G4368 and G4369 are designed using csu-15 gene orders, so
The precursor-gene of csu-15 artificial mi RNAs is synthesized with two-wheeled PCR afterwards, and the precursor-gene is building up to pC1300-Ubi-Nos and is carried
On body, recombinant plasmid pUbi-ami-csu15 is obtained, 11 are spent with the recombinant plasmid transformed rice material, by the transgenosis of acquisition
Rice material be named as paddy rice ZH11-csu15, Oryza sativa L.ZH11-csu15, delivered on November 29th, 2013
Chinese Wuhan Wuhan Universitys China typical culture collection center preservation, its preserving number is CCTCC NO：P201307；
Wherein：The nucleotide sequence of csu-15 artificial mi RNA precursor-genes such as SEQ ID NO：Shown in 3；
The following institute of nucleotide sequence of described 15-I, 15-II, 15-III, 15-IV, G4368 and G4369 primer sequence
Show：
15-I agTATAACTATAGATAGTACAGTcaggagattcagtttga（5′–3′）；
15-II tgACTGTACTATCTATAGTTATActgctgctgctacagcc（5′–3′）；
15-III ctACTGTTCTAACTATAGTTATAttcctgctgctaggctg（5′–3′）；
15-IV aaTATAACTATAGTTAGAACAGTagagaggcaaaagtgaa（5′–3′）；
G-4368CTGCAAGGCGATTAAGTTGGGTAAC（5′–3′）；
G-4369GCGGATAACAATTTCACACAGGAAACAG（5′–3′）；
Described recombinant plasmid pUbi-ami-csu15 contains SEQ ID NO：Csu-15 artificial mi RNA precursors shown in 3
Gene；
The PCR method is as described below：
Entered using primer combination G-4368+15-II, 15-I+15-IV and 15-III+G-4369 using plasmid pNW55 as template
Row first round PCR reacts, and reaction system is：10 × pfu Taq buffer2.0 μ l, dNTPs2.0 μ l, left and right each 0.2 μ of primer
L, pNW5510ng, pfu Taq0.2 μ l, sterilizing distilled water to 20 μ l；
Reaction condition：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃7min.
By PCR primer in 0.8% agarose gel electrophoresis, PCR primer is reclaimed after digging glue, 20 μ l sterilizing distilled waters are finally dissolved in；
The second wheel PCR is carried out with the first round PCR of recovery three kinds of PCR primers；
Second wheel PCR is combined using G-4368+G-4369 primers, and reaction system is：10 × Ex Taq buffer2.0 μ l,
DNTPs2.0 μ l, G-43680.2 μ l, G-43690.2 μ l, first round PCR primer mixed in equal amounts 1.0 μ l, Ex Taq
0.2 μ l, sterilizing distilled water to 20 μ l；
Reaction condition：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃7min；Final PCR
Product cloning is on carrier T pEASY-T3, then sequence verification；Contain csu-15amiRNA using BamH I and Kpn I digestions
The carrier T of precursor-gene, and the precursor-gene discharged is cloned on carrier pC5300-Ubi-tNos and obtains final expression vector
pUbi-ami-csu15。
The advantage of the invention is that：
1. the present invention uses RNAi technology, its pest-resistant mode does not have toxalbumin and the toxin for introducing and having harm to environment.
2. the amiRNA precursor-gene fragments of the present invention are small, no protein product, overexpression is difficult in transfer-gen plant
The metabolism burden of increase overexpression plant.
3. the exogenous gene sequence of the present invention derives from the endogenous tiny RNA of striped rice borer, and fragment very little only 21nt, target holds
Easily prediction, potential risks are smaller.
Brief description of the drawings
Sequence table SEQ ID NO：1 is striped rice borer tiny RNA csu-15 DNA sequence dna.
Sequence table SEQ ID NO：2 be the DNA sequence dna of the natural precursor of striped rice borer tiny RNA csu-15 predictions.
Sequence table SEQ ID NO：3 be the DNA sequence dna of striped rice borer tiny RNA csu-15 artificial mi RNA precursor-genes
Fig. 1：Striped rice borer tiny RNA bioinformatics is annotated and the pre- flow gauges of new microRNA.
Fig. 2：Expressions of the striped rice borer tiny RNA csu-15 in different development stage is detected by RT-PCR technology.
Fig. 3：It is plasmid vector pNW5 structural representations of the present invention
Fig. 4：It is initial carrier pC1300-Ubi-Nos structural representations of the present invention.
Fig. 5：It is the whole carrier that the present invention is built（Recombinant plasmid）PUbi-ami-csu15 structural representations.
Fig. 6：The in vitro stalk of transgenic paddy rice connects worm qualification result.In Fig. 6：The left side is to strip out in transgenic paddy rice stalk
Striped rice borer；The right is the striped rice borer that strips out in non-transgenic reference stalk.
Fig. 7：It is first time of the invention to connect worm qualification result.
Fig. 8：It is that the present invention connects worm qualification result for the second time.
Fig. 9：It is that third time of the invention connects worm qualification result.
Embodiment
Embodiment 1:The extracting of striped rice borer tiny RNA sample
The life cycle of striped rice borer includes four growth and development stages：Ovum, larva, pupa and adult.Wherein larval phase, is usual
It is divided into 5 ages.The sample that the present invention have collected 6 periods of growing of striped rice borer includes：Ovum, 1-2 instar larvaes, 3-4 ages children
Worm, 5 instar larvaes, pupa and adult.It is collected into after each period striped rice borer sample, is first put into liquid nitrogen container and preserves, carry sample is collected
It is stripped after neat using Trizol reagents, method is as follows：
1) the striped rice borer sample for weighing 0.1g is pulverized in liquid nitrogen.
2) add the rapid sample powder by after grinding of 5ml Trizol reagents to mix, be divided in 5 1.5ml RNAase-
In free centrifuge tube.
3) room temperature places several minutes and is melted into liquid completely to mixture.
4) 4 DEG C of 5000g centrifugation 10min, and shift supernatant into new 1.5ml RNAase-free centrifuge tube, this
Step can remove unnecessary tissue hybrid.
5) add the chloroform of 200 μ l frosts and shake 15s.
6) 4 DEG C of 5000g centrifugation 10min, and shift supernatant into new 1.5ml RNAase-free centrifuge tube
7) repeat step 5 and 6 is continued.
8) 500 μ l isopropanols are added in the supernatant being transferred out of, are well mixed, -20 DEG C of frosts are stayed overnight.
9) 4 DEG C of 5000g centrifugation 30min, abandon supernatant.
10) the 500 μ l75% ethanol desalinization of soil by flooding or leaching are added.
11) 4 DEG C of 5000g centrifugation 5min, abandon supernatant.
12) centrifugation lid is opened, air drying RNA, the time is no more than 10min.
13) the treated ddH2O of 30-50 μ l DEPC are added.
14) flicking makes RNA quickly dissolve, and can dissolve RNA in 65 DEG C of water-baths.
Embodiment 2:The recovery of striped rice borer tiny RNA sample and Solexa sequencings
The RNA sample in 6 periods of striped rice borer reclaims below 80nt small RNA fragments by gel electrophoresis, then by recovery
Small RNA fragments connection special 3 ' end connectors and 5 ' end connectors.Reverse transcription is carried out using 3 ' end primers, obtained product is again with 5 '
Tiny RNA high-flux sequence is carried out with after the primer PCR amplification at 3 ' ends.
It is sequenced by Solexa, each sample obtains 1 × 108 or so small fragment data volume, obtains dividing after these data
Analyse flow such as Fig. 1 institutes.Obtained fragment sequence is removed into 5 ' and 3 ' end connectors first, and removes low-quality sequence, is chosen
Go out the fragment of 18-30nt length.The fragment obtained after processing is compared with Genbank databases, to what is guarded in 6 samples
The tiny RNAs such as rRNA, scRNA, snoRNA, snRNA, tRNA, piRNA are annotated, then are compared with miRBase databases,
The conservative miRNA of annotation.After the conservative tiny RNA of annotation is excluded, the remaining tiny RNA combination striped rice borer not being accredited out
Group information and Mireap softwares is transcribed to predict new striped rice borer miRNA.
MiRNA derives from pri-miRNA precursors, is recognized after pri-miRNA is transcribed out in nucleus by Drosha
And pre-miRNA is cut into, and by Exportin-5 transporte to cells matter（Kurreck2009）.Therefore, striped rice borer is transcribed
There is pre-miRNA sequence information in group database.First the small RNA fragments of sequencing are navigated in the sequence of transcript profile, length
It is preset as the general length of miRNA precursors, about 70nt（Lee et al.2003）, two grades that this section of precursor sequence is then predicted again
Can mechanism, see its hairpin structure two-arm match condition, form hairpin structure and the free energy of structure, and final selection one is fitted
The sequence of conjunction is contrasted the small RNA fragments of sequencing with it as precursor, is chosen the high sequence of matching, expression quantity completely and is made
For the special miRNA of the ripe bodies of new miRNA of prediction, that is, species.
Predicted with striped rice borer transcript profile database while miRNA, also using the transcript profile of striped rice borer midgut tissue come
Special miRNA is predicted, so predicted with two kinds of striped rice borer transcript profile databases come special miRNA it is common
As a result, it is not only truer in confidence level, and some special miRNA for being present in midgut tissue can be gone out with tentative prediction, because
For these miRNA precursor in striped rice borer midgut tissue, it is formed in midgut tissue, its act on target gene be also accordingly present in
In midgut tissue.
Prediction more than and it is assumed that obtain tiny RNA sequence csu-15, its sequence such as sequence table SEQ ID NO：1 institute
Show, it organizes all to be predicted to be new miRNA in transcript profile and middle intestines transcript profile entirely in striped rice borer, therefore primarily determines that csu-15 is deposited
It is striped rice borer midgut tissue, its natural precursor gene order such as sequence table SEQ ID NO：Shown in 2.
Embodiment 3:The checking of tiny RNA cus-15 expression
The new miRNA csu-15 of striped rice borer gone out by high-flux sequence and Bioinformatics Prediction, in addition it is also necessary to pass through stem ring
RT-PCR（Chen et al.2005）And the method for experiment such as RT-PCT products are sequenced again to verify its authenticity and standard
True property.Its method is as follows.
According to the candidate's miRNA results predicted, special reverse transcription primer and PCR primer are designed, involved draws
Thing is as shown in table 1.
Primer sequence needed for the stem ring RT-PCR of table 1
Remove the genomic DNA in sample with DNase I processing first, then with special reverse transcription primer 15-specific-
R carries out reverse transcription respectively to 6 samples of striped rice borer, and system is as follows：
RNA sample：1.0μg
DNase I：0.5μl
10×DNase I buffer 1.0μl
Mend the ddH of DEPC processing2O to 10 μ l
37 DEG C of reaction 15min.
65 DEG C of water-baths inactivate 10min.
2min on ice is placed on after inactivation, is somewhat centrifuged.
Add Reverse Transcription：
Mend the ddH of DEPC processing2O to 20 μ l
16 DEG C of water-bath 30min.
38 DEG C of water-bath 30min.
70 DEG C of water-baths inactivate 10min.
The product that reverse transcription is obtained carries out RT-PCR with PCR primer and universal primer, and reaction system is
ddH2O moisturizings are to 20 μ l
PCR conditions：
RT-PCR results have expression as shown in Fig. 2 csu-15 small fragments are interim in striped rice borer 6, and in larva
Phase and pupa time expression are high compared with ovum phase and adult stage.
Use pEASY-T3Cloning Kit（Purchased from Quan Shi King Companies）TA clones are carried out to its stem ring RT-PCR product,
And TA plasmids are sequenced.Method is as follows：
1) 3 μ l are drawn and 1 μ l pEASY-T3Cloning vector are added, are well mixed in room temperature reaction 5min, rapidly
It is placed on ice.
2) completely reacted mixture is added in 50 μ l competent escherichia coli cells, be placed on ice after gently mixing
25min。
3) 42 DEG C of water-bath heat shock 30s, are immediately placed in 2min on ice.
4) 250 μ l LB resuscitation fluids are added, and Soviet Union 1h is replied immediately in 37 DEG C of shaking table 170rpm.
5) recovery product is uniformly applied on the LB culture dishes containing ampicillin.
6) 37 DEG C of overnight incubations.
After TA clones finish, the picking monoclonal on the LB culture dishes containing ampicillin is put into 2ml（100mg/L）
Overnight incubation in amicillin resistance LB, and carry out plasmid extraction.Digestion detection is carried out to the plasmid extracted, insertion is selected
The positive plasmid sequencing of RT-PCR product fragments.Sequencing result shows, the sequence for the csu-15 that stem ring RT-PCR is obtained with it is high
The result of flux sequencing is completely the same.
Embodiment 4:The structure of the artificial mi RNA expression vector of csu-15 fragments
The acquisition of artificial mi RNA expression vector is from Warthmann etc.（Warthmann,Chen et al.2008）Report
The driving of corn Ubiquitin promoters special miRNA sequence, the building mode of the present embodiment is consistent with its, with plasmid
pNW55（See Fig. 3, provided by German Ma Pu biological developments research institute Detlef professors Weigel）For template, pass through overlap-extension PCR
PCR method amplifies DNA fragmentation.Specific primer is as shown in table 2, altogether comprising 6, wherein 2 are G4368 and G4369
Universal primer, 4 are respectively primer 15-I, 15-II, 15-III and 15-IV in addition.
Primer sequence needed for the amiRNA expression vector establishments of table 2
First round PCR reaction template is pNW55, and G-4368+15-II, 15-I+15-IV and 15- are combined using primer
III+G-4369, reaction system is：
Mend ddH2O to 20 μ l.
PCR conditions are：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30cycles；72℃7min.
The pfu Taq that PCR is used are pfu polymerase（New England Biolabs, the U.S.）, to PCR primer
Detected through gel electrophoresis is carried out, and with QIAquick Gel Extraction Kit（QIAGEN, Germany）Kit reclaims PCR productions
Thing, is finally dissolved in 20 μ l ddH2O, the product being recovered to is mixed into a pipe and as second of PCR template.
Second wheel PCR uses G-4368 and G-4369 universal primers, and reaction system is as follows：
Mend ddH2O to 20 μ l
PCR conditions are：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30cycles；72℃7min.
The Taq that PCR is used is Ex polymerase（TaKaRa）, detected through gel electrophoresis is carried out to PCR primer and to its piece
Section uses pEASY-T3Cloning Kit（Quan Shijin）TA clones are carried out, and correct matter is verified with enzymatic cleavage methods to TA plasmids
Grain is sequenced, and the correct plasmid of selection sequencing is connected into the previous structure in this laboratory with target fragment under BamH I and Kpn I digestions
The plasmid vector pC1300-Ubi-Nos built（Fig. 4, Chen et al.2013）, build the whole carrier for forming the present invention（Or again
Group plasmid）pUbi-ami-csu15（Structure is shown in Fig. 5）.
Embodiment 5:The genetic transformation of paddy rice
It is transformed into the whole expression vector pUbi-ami-csu15 built by agrobcterium-mediated transformation
Spent in rice varieties in 11, the rice material of the transgenosis of acquisition is named as paddy rice ZH11-csu15, Oryza sativa
L.ZH11-csu15, Chinese Wuhan Wuhan Universitys China typical culture collection center guarantor is delivered on November 29th, 2013
Hide, its preserving number is CCTCC NO：P201307；
Agrobacterium-mediated genetic transformation is comprised the following steps that：
（1）Callus is induced：
Say and spend 11 rice paddy seeds to shell processing in maturation, then successively with 75% Ethanol Treatment 1 minute, 0.15% chlorination
Mercury the surface of the seed is sterilized 20 minutes.Washing seed is steamed with sterilizing is single 4-5 times, and seed is uniformly placed on inducing culture.Inoculation
Culture medium afterwards is positioned over dark room and cultivated 4-6 weeks, 28 ± 1 DEG C of temperature.
（2）Callus subculture
The embryo callus subculture of picking glassy yellow, consolidation and relatively dry, is put on subculture medium and is cultivated 3 weeks in darkroom,
28 ± 1 DEG C of temperature.
（3）Preculture
The embryo callus subculture of picking consolidation and relatively dry, is positioned on pre-culture medium under dark condition and cultivates 4 days, temperature 28
±1℃.（4）Agrobacterium is cultivated
The preculture Agrobacterium 2 days on the LA culture mediums with correspondence resistance selection, temperature is 28 ± 1 DEG C.By Agrobacterium
Move in suspension medium, with 28 DEG C on shaking table, 200rpm CMC model 2-3h.
（5）Agrobacterium is infected
The callus of preculture is transferred in the bottle of sterilizing.The suspension of Agrobacterium is adjusted to OD600=0.3 or so.More
Wound soaks 10min in agrobacterium suspension.Blotted on transfer callus to sterilized filter paper, be then placed into co-culturing on base
Cultivate 3d, 19-20 DEG C of temperature.
（6）Callus is washed and selection culture
Rear callus is infected with sterilizing water washing 7-8 times, is then immersed in the aqua sterilisa of the carbenicillin containing 400mg/L
Middle 30min.Transfer callus selects to cultivate 3 times to after blotting on filter paper sterilize, in transfer callus to Selective agar medium, every time
For 2 weeks.
（7）Differentiation
Kanamycin-resistant callus tissue is transferred into pre- differential medium to cultivate at dark 7 days, 26 ± 1 DEG C of temperature.The pre- differentiation training of transfer
Foster callus is cultivated, 26 ± 1 DEG C of temperature to differential medium under illumination.
（8）Take root
Cut the root produced during differentiation；It is then transferred on root media under illumination and cultivates 2-3 weeks, temperature 26 ±
1℃.（9）Transplant
By the remaining medium wash clean on root, the seedling with good root system is transferred to greenhouse, while initial
Moisture moistening is kept in several days.
The patent document that the culture medium and its compound method of the present embodiment are authorized referring to Hua Zhong Agriculture University：The patent No.
2011100832269, authorized announcement date 2013 year 08 month 21 days.
Embodiment 6:Transgenosis regenerates the positive detection of rice plant
After regenerated transgenic seedling is transplanted 3 weeks, its blade is taken respectively, extracts DNA（Specific method is seen below）.Drawn using hygromycin
Thing enters performing PCR positive detection.Hygromycin primer sequence is：Hpt-F5 '-AGAATCTCGTGCTTTCAGCTTCGA-3 ' and Hpt-
R5’-TCAAGACCAATGCGGAGCATATAC-3’。
（1）Rice leaf miniprep dna is extracted：
1) 3-5cm young leaflet tablet is taken；
2) blade is worn into homogenate on sample grinding machine, water-bath in 800 μ l 15 × CTAB buffer solutions, 65 DEG C of water-baths is added
30min, during which shakes up once per 5min；
3) volume ratio 24 of equivalent is added:1（Chloroform/isoamyl alcohol）, light and slow reverse mixing 5-10min；
4) 12000rpm centrifuges 10min；
5) 300 μ l supernatant is drawn, is transferred them in another clean centrifuge tube, -20 DEG C of precoolings of 2 times of volumes are added
Absolute ethyl alcohol, -20 DEG C standing 30min；
6) 12000rpm centrifuges 15min；
7) supernatant is abandoned, 500 μ l 75% ethanol is added, washing is mixed up and down；
8) 12000rpm centrifuges 5min;
9) supernatant is abandoned, in being dried up on superclean bench, the ddH of 100 μ l sterilizings is added2O, room-temperature dissolution.
PCR reaction systems：
Use ddH2O moisturizings are to 20 μ l
PCR conditions are：95 DEG C of 5min, 95 DEG C of 5s, 59 DEG C of 30s, 72 DEG C of 1min30cycles；72℃7min.PCR primer is led to
Cross agarose gel electrophoresis detection, the positive plant of picking.Resulting positive T0For obtaining T after plant breeding1For plant, so
Afterwards to T1Positive detection is carried out again for plant.Extract positive T1For the leaf tissue RNA of plant, detected by stem ring RT-PCR
The expression quantity of purpose fragment, RNA method for extracting and stem ring RT-PCR detection is same as Example 3.To the side by stem ring PCR
Method filters out the higher plant of expression quantity and does further detection.
Embodiment 7:The worm that connects of transfer-gen plant is identified
The T for obtaining high expression quantity is detected by stem ring RT-PCR1For transfer-gen plant, carry out striped rice borer and connect worm identification.Connect
The specific method of worm identification is as follows：
（1）Take the transfer-gen plant after jointing and original kind（Non-transgenic）In spend 11 rice plant stalks；
（2）The 5 rhizome stalks that each family takes length consistent are placed in a clean pipe of taking root, and inoculation 20 is in the same size
The Chilo spp larvae just hatched, is taken root the mouth of pipe with ParafilmTM, and each family repeats to do 5 pipes；
（3）Raised 7 days for 28 DEG C in illumination box；
（4）Stalk is peeled off, Chilo spp larvae is taken out, statistics carrys out the average weight of striped rice borer insect in each family stalk.
The result for connecing worm identification shows that the average weight of striped rice borer compares feeding pair in transgenic paddy rice material after being fed with 7 days
According to it is middle spend 11 compared to significantly decreasing（Fig. 6）.We have carried out three repetition experiments different in the period of.Connect for the first time
Worm as shown by data, compared with spending 11 in control, the striped rice borer average weight in transgenic paddy rice stalk declines 33.3%（P=
0.0278, Fig. 7）, with significant difference.Worm as shown by data is connect for the second time, compared with spending 11 in control, in transgenic paddy rice stalk
Striped rice borer average weight decline 57.1%（P=0.0162, Fig. 8）, with significant difference.Third time connects worm as shown by data, and right
11 are spent to compare according in, the striped rice borer average weight in transgenic paddy rice stalk declines 38.3%（P=0.0001, Fig. 9）, with extremely aobvious
Write difference.
It is above-mentioned to connect worm experiment respectively in different period progress, it is that csu-15 pieces are expressed in feeding in single independent experiment
Section rice plant after striped rice borer worm downward trend is basically identical again, illustrate that csu-15 fragments are right after being overexpressed in rice plant
The growth of striped rice borer has significant supression to act on, with certain insect resistant effect.
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Claims (2)
1. nucleotides sequence list SEQ NO：Tiny RNA csu-15 genes shown in 1 are in the growth of rice grub striped rice borer is suppressed
Using.
2. application as claimed in claim 1, its feature comprises the following steps：
Utilize csu-15 gene orders design amplimer 15-I, 15-II, 15-III, 15-IV, G4368 and G4369, Ran Houyong
Two-wheeled PCR synthesizes the precursor-gene of csu-15 artificial mi RNAs, and the precursor-gene is building up into pCAMBIA5300-Ubi-tNos
On carrier, recombinant plasmid pUbi-ami-csu15 is obtained, 11 is spent with the recombinant plasmid transformed rice material, obtains transgenosis water
Rice (Oryza sativa L.) material ZH11-csu15；
Wherein：The nucleotide sequence of csu-15 artificial mi RNA precursor-genes such as sequence table SEQ ID NO：Shown in 3；
Wherein：The nucleotide sequence of 15-I, 15-II, 15-III, 15-IV, G4368 and G4369 primer sequence is as follows：
15-I agTATAACTATAGATAGTACAGTcaggagattcagtttga；
15-II tgACTGTACTATCTATAGTTATActgctgctgctacagcc；
15-III ctACTGTTCTAACTATAGTTATAttcctgctgctaggctg；
15-IV aaTATAACTATAGTTAGAACAGTagagaggcaaaagtgaa；
G-4368 CTGCAAGGCGATTAAGTTGGGTAAC；
G-4369 GCGGATAACAATTTCACACAGGAAACAG；
Wherein recombinant plasmid pUbi-ami-csu15 contains sequence table SEQ ID NO：The csu-15 artificial mi RNAs of sequence shown in 3
Precursor-gene；
Above-mentioned PCR method is as described below：
The is carried out by template of plasmid pNW55 using primer combination G-4368+15-II, 15-I+15-IV and 15-III+G-4369
One wheel PCR reacts, and reaction system is：10 × pfu Taq buffer 2.0 μ l, dNTPs 2.0 μ l, left and right each 0.2 μ l of primer,
The μ l of pNW55 10ng, pfu Taq 0.2, sterilizing distilled water to 20 μ l；
Reaction condition：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30cycles；72℃7min；
By PCR primer in 0.8% agarose gel electrophoresis, PCR primer is reclaimed after digging glue, 20 μ l sterilizing distilled waters are finally dissolved in；With
The first round PCR of recovery three kinds of PCR primers carry out second and take turns PCR；
Second wheel PCR is combined using G-4368+G-4369 primers, and reaction system is：The μ l of 10 × Ex Taq buffer 2.0,
DNTPs 2.0 μ l, G-43680.2 μ l, G-4369 0.2 μ l, the μ l of 1.0 μ l, Ex Taq of first round PCR primer mixed in equal amounts 0.2,
Distilled water sterilize to 20 μ l；
Reaction condition：95℃2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30cycles；72℃7min；Final PCR primer
It is cloned on carrier T pEASY-T3, then sequence verification；Contain csu-15amiRNA precursors using BamH I and Kpn I digestions
The carrier T of gene, and the precursor-gene discharged is cloned on carrier pC5300-Ubi-tNos and obtains final expression vector
pUbi-ami-csu15。
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