CN102559666B - Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof - Google Patents

Abstract

The present invention relates to a plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof. The invention firstly designs oligonucleotides with excellent silencing effect according to the nucleic acid sequence of a gene silencing suppressor of a plant virus infecting plants. When the virus infects a plant, these oligonucleotides transfected into the plant can form miRNA in the plant which specially binds to the corresponding parts of mRNA (massager RNA) of the virus gene silencing suppressor to affect the transcription of the mRNA, thereby achieving virus inhibitory effect. In addition, oligonucleotides of the invention are optimized and designed on the basis of locating the most conservative positions after comparing the virus gene silencing suppressors derived from different virus subtypes, the oligonucleotides designed based on these positions has broad-spectrum resistant effect on related viruses after being introduced into the plant.

Description

The artificial mi RNA and structure and the purposes that suppress plant virus

Technical field

The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to suppress artificial mi RNA and structure and the purposes of plant virus.

Background technology

Chinese cabbage group mainly comprises balling class Chinese cabbage (Brassica campestris L.ssp.pekinensis) and balling class Plantula Brassicae chinensis (Brassica campestris L.ssp.chinensis) not.Plantula Brassicae chinensis is called for short green vegetables, and the north claims rape, its strong adaptability, and growth is fast, and output is high, and nutrition is good, and consumption occupies first of all kinds of vegetables, is a kind of popular vegetables that generally cultivate each province, China Yangtze valley.Plantula Brassicae chinensis kind and various in style, vegetative period is short, wide adaptability, high yield, the saving of labor, easily plant, but the anniversary production and supply.Product is fresh and tender, nutritious, by consumers in general are liked.Its annual production accounts for the 30%-40% of vegetables ultimate production, to supplementing vegetables dull seasons, the stable market supply contribution of the anniversary of realization is very large.Chinese cabbage and Plantula Brassicae chinensis characteristic are all the cool property of happiness, all can produce throughout the year, and the growth optimum temperuture is 15～20 ℃.In recent years, in order to adapt to the needs in market, intensive culture is the principal feature that Chinese cabbage group is produced.In order to guarantee Chinese cabbage group balanced production and supply throughout the year, Plantula Brassicae chinensis often needs to be produced in different ways throughout the year.

In brassicaceous vegetable, Chinese cabbage, Plantula Brassicae chinensis, rape, radish, wild cabbage etc. all can be subject to virus infection, cause virus disease, and wherein Chinese cabbage virus disease is one of three the most serious large diseases of Chinese cabbage group harm.The main pathogen virus that can infect Chinese cabbage and cause serious virus disease to occur has three kinds, be Brassica 2 et 4 (TuMV, Turnip mosaic virus.), cucumber mosaic virus (CMV, Cucumber mosaic virus.) and tobacco mosaic virus (TMV) (TMV, Tobacco mosaic virus.).TuMV is a kind of curve strip virus, and long 680-750nm can exist on the vegetables such as brassicaceous vegetable and spinach, leaf mustard and weeds, and its longevity in vitro only has 2-4 days, dilution end point be 1000-10000 doubly, fatal temperature 55-60 ℃.Incubation period temperature influence in seedling, when on the low side the incubation period long, short incubation period when higher, between 9-14 days, carry out the juice propagation by aphid as amboceptor.In the North, Northeast China area, when open country does not have Chinese cabbage and other hosts to grow, in the Chinese cabbage of storing sth. in a cellar and other brassicaceous vegetables and their kind strain, can there is virus to exist, become Second Year during the warmth of spring, all the flowers bloom the first source of infecting of field of vegetables in season.The area that greenhouse and plastic greenhouse are arranged, become first the infect source of virus with the overwintering host circulating propagation.Evergreen all the year round southern area, there is not winter issue in virus, and circulation is infected throughout the year.Mainly in winter in spring, produce Plantula Brassicae chinensis in the past, and started to take various planting type plantation Plantula Brassicae chinensiss in more hot summer and autumn, undoubtedly within its breeding time, especially spring Mo, summer and early autumn tended to be subject to the infringement of virus disease.High temperature season cultivation Plantula Brassicae chinensis can go on the market at 20 days later in batches, but obstacle by a high temperature often makes the Plantula Brassicae chinensis plant produce virus disease, usually causes yielding poorly, poor quality, and market value raises up, and supply falls short of demand, can not meet resident's consumers demand.

Chinese cabbage and Plantula Brassicae chinensis originate in China, external less to the breeding research of Chinese cabbage group, and the variety resistance of introducing from Japan, Korea S and China Taiwan is poor, are not suitable in China's growth in summer and promote.The kind of domestic institute seed selection is mainly the autumn culture kind, and Chinese cabbage group viral diseases gene pool is narrow, and disease-resistant breeds of Chinese cabbage seed selection only is confined to the screening between the Chinese cabbage material, and the variety resistance of selecting is not ideal enough.In view of the foregoing, China breeding man adopts traditional breeding method extensively to carry out the seed selection work of disease-resistant Chinese cabbage group kind, introduces disease-resistant gene, enlarges the source resource approach, to a certain degree improve the Chinese cabbage group resistance against diseases, on producing, brought into play effect.Yet viral variability own is strong, region is different can produce many new physiological strains, and the disease-resistant variety of releasing is in the past lost disease resistance to a certain extent.And the region restriction is arranged, disease tolerant variety extensively can not be generalized to other areas.Therefore the genesis Changing Pattern of cabbage vegetable virus disease symptoms carried out to deep research, set up easy handling, result is stable, and the viral diseases screening method with extensive generalization that efficiency is higher and technology are that the solution task is needed in the work of Chinese cabbage group breeding for disease resistance badly.Belong to quantitative character with the disease-resistant closely-related proterties of Chinese cabbage, it is very difficult that the genotype of its quantitative character is selected.For molecular breeding, this difficulty not only shows that the DNA marker that can utilize with assisted Selection is few, and QTL (Quantitative Traits Loci, quantitative trait locus) is at number with to use variation larger.Therefore, in the work of Chinese cabbage gene order-checking, not yet complete, in functional genome research situation in the ascendant, Genetic breeder need to be found a kind of quick, responsive, efficient, each proterties and DNA Technique of Qualitative Analysis on the plant level, and the quantitative analysis tech of phenotype and changes in gene expression on the plant level, in order to the Chinese cabbage group breeding for disease resistance, study.

Molecular biology research development in recent years rapidly, genetic engineering technique has been one of the most far-reaching great Progress & New Products of impact since the mid-90 in 20th century, is to melt the new technology that height that microtronics, biology, physics, chemistry, computer science be integrated intersects.At present, gene engineering has been widely used in many fields such as drug screening, agricultural, medical diagnosis on disease and treatment, the evaluation of Chinese medicine species, judicial expertise, Food Hygiene Surveillance, environment measuring, national defence.The report of applying in plant about gene engineering is also many, mainly concentrates on the aspects such as Arabidopis thaliana, strawberry, morning glory.

The kind of the viroses of plant is a lot, distributes very wide, serious to the harm of farm crop.Usually, the symptom of the viroses of plant is that plant forms is abnormal, and plant-growth and growth present systematicness and be obstructed.Take Chinese cabbage as example, and when Seedling Stage is injured, the lobus cardiacus initial stage produces bright arteries and veins, and sick leaf shrinkage becomes fragile, the distortion deformity, and the grave illness strain is obviously stunt, and does not wrap the heart, and blade is firmly crisp, shrinkage is agglomerating, and root system is undeveloped.For a long time, virus is that how to cause the systematicness of plant to infect be puzzlement plant protection scholar's a science difficult problem always.What is interesting is, the phenotype of the mutant such as these virus disease symptoms and model plant Arabidopis thaliana dicer-likel (dcll), hyponastic leaves (hyll), serrate (se) and agonautel (agol) is closely similar.In higher plant, DCL1, HYL1 and SE albumen participate in the biological processing of miRNA, and AGO1 albumen is the key protein of gene silencing (gene silencing).The function of these albumen is the gene silencing reactions that keep in plant materials, guarantees that plant grows normally.Genetically deficient or sudden change all can affect the silent reaction of downstream target gene, cause the form reaction that is similar to the plant virus disease symptoms.Generally, normal plant gene silencing has the antiviral effect with alleviating virus harm.Research discovery in recent years, the similarity of virus disease symptom and miRNA route of synthesis mutant is associated on molecular level.Exist inhibition (suppressor) of RNA silence in viral genome, this suppresses the function that son can disturb plant DCL1, HYL1, SE or AGO1 albumen or relevant miRNA, makes the plant gene silencing approach impaired, and causes a series of form reactions.

MiRNA (21-24nt) is the messenger molecule between genome and protein group, exercises in vivo multiple adjusting function.In plant, miRNA mainly participates in the processes such as genome stabilization, gene silencing and biodefense.Under the acting in conjunction of DCL1, HYL1 and HEN1, precursor miRNA (pre-miRNA) produces ripe miRNA through shearing, and ripe miRNA is at RISC (miRNP/RNA-induced silencing complex, miRNP/RISC) effect is lower can combine with the complementary sequence on target gene mRNA, target gene mRNA is degraded and suppress its expression.AGO1 albumen participates in the mechanism of RISC silencing complex.Up-to-date statistic data shows, Arabidopis thaliana has 46 miRNA families and 117 miRNA genes [Small RNA-directed epigenetic natural variation in arabidipsis thaliana.PLOS genetics (2008) 4:e1000056].Along with the raising of miRNA diagnostic techniques, also will there is new miRNA constantly to be found.

In plant materials, pre-miRNA produces ripe miRNA through shearing, and the latter is with the degrade mRNA of target gene of the mode of sequence complementation.Utilize the characteristic of Mirnas of plant processing, some laboratory has built the artificial mi RNA carrier of viral diseases.Its principle is, viral silencing suppressors for ripe miRNA (as P1/HC-Pro) fragment endogenous in plant pre-miRNA sequence is replaced, build the expression vector of pre-miRNA, proceed to plant under the mediation of Agrobacterium, produce artificial mi RNA in plant materials, the mRNA of degraded virogene silencing suppressors, remove the blocking effect of virogene silencing suppressors to plant gene silencing, the purpose that can't occur to reach the virus disease symptom.The New Policy of this viral diseases has broad application prospects in Resistant breeding.2006, Niu etc. utilized the pre-miR165 carrier of P1/HC-Pro to create the transgenic plant of Arabidopis thaliana, make P1/HC-Pro artificial mi RNA successful expression [Niu, Q.W., Lin, S.S., Reyes, J.L., Chen, K.C., Wu, H.W., Yeh, S.D.and Chua, N.H. (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance.Nat Biotechnol, 24,1420-1428].This miRNA causes the mRNA degraded of P1/HC-Pro, and the plant of screening shows stronger resistance to the strain of some TuMV and TYMV virus.At present, the research of artificial mi RNA viral diseases also rests on the model plant Arabidopis thaliana, and antiviral object is limited in some viral America strains.On the other hand, the factor that affects artificial mi RNA biological processing, maturation and target gene silence is a lot, need to be improved and optimize whole artificial mi RNA system, to improve the efficiency of transgenic plant viral diseases.The more important thing is how the artificial mi RNA system is applied to farm crop and cash crop, make it to become the effective way of Resistant breeding.

For a long time, the breeding scholar cultivates the improved seeds of antiviral proterties by the method for sexual hybridization, and the cultivation scholar manages to alleviate the harm of virus disease in order to produce the quality vegetables product.Yet, due to the restriction of discipline development level and research means, also, due to the genetic phenomenon of virus disease complicated symptoms, people are difficult to judge according to traditional Heredity theory the genetic development of these symptoms.Be reflected on breeding practice, thremmatology man is usually perplexed by complicated Antiviral Mechanism.Improve the progress of Chinese cabbage Anti-virus Disease Breeding, cultivate disease-resistant variety of new generation, just must the new Anti-virus Disease Breeding new technology of development.

Summary of the invention

The object of the present invention is to provide the artificial mi RNA and structure and the purposes that suppress plant virus.

In a first aspect of the present invention, oligonucleotide a kind of separation or artificial constructed is provided, described oligonucleotide is selected from:

(i) SEQ ID NO:1-SEQ ID NO:4 arbitrary shown in the oligonucleotide of sequence; Or

(ii) with the oligonucleotide of the arbitrary sequence complementation (i) limited.

In another aspect of this invention, provide a kind of construction, described construction contains described oligonucleotide, and it can express the corresponding miRNA of described oligonucleotide after importing vegetable cell, tissue or organ.

In another preference, in described construction, contain:

Be selected from the sequence (corresponding to cucumber mosaic virus (CMV)) of SEQ ID NO:1 or SEQ ID NO:3; With

Be selected from the sequence (corresponding to Brassica 2 et 4 (TuMV)) of SEQ ID NO:2 or SEQ ID NO:4.

In another preference, described construction is expression vector.

In another preference, described expression vector is the pBSK carrier.

In another preference, described construction contains at least one group of structure shown in formula I:

Seq _forward-X-Seq _oppositelyformula I;

In formula I,

Seq _forwardfor described oligonucleotide sequence;

Seq _oppositelyfor with Seq _forwardbasically complementary oligonucleotide sequence;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _oppositelynot complementary.

In another preference, the structure shown in formula I, after proceeding to vegetable cell, forms the secondary structure shown in formula II:

formula II,

In formula II, Seq _forward, Seq _oppositelywith stating as defined above of X,

|| be illustrated in Seq _forwardand Seq _oppositelybetween complementary relation basically.

In another preference, the length of described intervening sequence is 5-500 Nucleotide (nt); More preferably 10-300nt; More preferably 50-200nt; More preferably 80-150nt; More preferably 120-140nt, as 130nt.

In another preference, described construction contains:

(Seq _{forward 1}-X-Seq _{reverse 1}) (Seq _{forward 2}-X-Seq _{reverse 2}) formula III;

In formula III,

Seq _{forward 1}for being selected from the sequence of SEQ ID NO:1 or SEQ ID NO:3;

Seq _{forward 2}for being selected from the sequence of SEQ ID NO:2 or SEQ ID NO:4;

Seq _{reverse 1}and Seq _{reverse 2}for respectively with Seq _{forward 1}and Seq _{forward 2}basically complementary oligonucleotide sequence.

Structure shown in formula III, after proceeding to vegetable cell, forms two loop-stem structures.

In another aspect of this invention, also provide the purposes of described oligonucleotide, for the preparation of antiviral reagent; Or for the preparation of the reagent that improves the plant virus resistance ability.

In another aspect of this invention, also provide the purposes of described construction, for the preparation of antiviral reagent; Or for the preparation of the reagent that improves the plant virus resistance ability.

In another aspect of this invention, also provide a kind of method that improves the plant virus resistance ability, described method comprises step:

(a) provide described construction;

(b) construction described in (a) is imported in plant (comprising vegetable cell, tissue or organ).

In another preference, described construction is expressed the corresponding miRNA of described oligonucleotide after importing in plant, and described miRNA suppresses the expression of virogene; And then raising plant virus resistance ability.

In another preference, the described method that construction is imported to plant comprises:

(1) provide the Agrobacterium of carrying the described arbitrary construction of claim 2-6;

(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thereby make described construction proceed to vegetable cell or tissue or organ.

In another preference, described method also comprises:

(3) select vegetable cell or tissue or the organ that proceeds to described construction;

(4) vegetable cell in step (3) or tissue or neomorph are become to plant.

In another preference, described virus is: Brassica 2 et 4 (Turnip mosaic virus., TuMV) or cucumber mosaic virus (Cucumber mosaic virus., CMV).

In another preference, described virogene is selected from: the RNA silencing suppressor HC-Pro of Brassica 2 et 4; Cucumber mosaic virus RNA silencing suppressor 2b.

In another aspect of this invention, also provide a kind of plant, the corresponding miRNA of the described oligonucleotide of its expression in vivo; Or comprise described construction in its body.The present invention also comprises offspring's (filial generation) plant of described plant.

In another aspect of this invention, also provide a kind of miRNA, a kind of miRNA, its be SEQ ID NO:37-SEQ ID NO:40 arbitrary shown in the miRNA of nucleotide sequence.

In another aspect of this invention, also provide a kind of vegetable cell or tissue (described vegetable cell or be organized as the non-plant reproductive material), it is to be selected from lower group:

(a) with described construction, transform or the cell of transfection; Or

(b) cell that contains described miRNA.

Other side of the present invention, due to the disclosure of this paper, is apparent to those skilled in the art.

The accompanying drawing explanation

Fig. 1, carrier pRS300 plasmid map.

Fig. 2, artificial mi RNA clone schematic diagram.

The HC-Pro sequence contrast of the Chinese cabbage TuMV of Fig. 3, different sources.

The 2b sequence contrast of the Chinese cabbage CMV of Fig. 4, different sources.

The secondary structure of Fig. 5, HC-Pro sequence prediction.

The secondary structure of the 2b sequence prediction of Fig. 6, CMV.

The RT-PCR principle schematic of Fig. 7, miRNA.

Fig. 8, transfer-gen plant are expressed the RT-PCR electrophorogram of miRNA.Wherein, swimming lane amiR2b1-1, amiR2b1-2, amiR2b1HP1-1, amiR2b1HP1-2, for carry out the electrophoresis result of the amplified production of pcr amplification with the CMV-2b1RT-PCR primer, repeat 2 times; Swimming lane amiR2b2-1, amiR2b2-2, for carry out the electrophoresis result of the amplified production of pcr amplification with the CMV-2b2RT-PCR primer, repeat 2 times; Swimming lane amiRHP1-1, amiRHP1-2, amiR2b1HP1-1, amiR2b1HP1-2, for carry out the electrophoresis result of the amplified production of pcr amplification with the TuMV-HcPro1RT-PCR primer, repeat 2 times; Swimming lane amiRHP2-1, amiRHP2-2, for carry out the electrophoresis result of the amplified production of pcr amplification with TuMV-Hc Pro2RT-PCR primer, repeat 2 times.

Embodiment

The inventor, through extensive and deep research, first according to the nucleotide sequence of RNA silencing suppressor in the virus that infects plant, has designed the oligonucleotide of reticent effect excellence.After these oligonucleotide import in plant materials, when the virus infection plant, can in plant materials, form miRNA, thereby specific binding is to the corresponding site of the mRNA of virogene silencing suppressors, affect the Transcription of mRNA, thereby reach the effect that suppresses viral.And, oligonucleotide of the present invention is based on the virogene silencing suppressors in comparison different virus hypotype source and the most conservative position that navigates to optimizes and design, oligonucleotide based on these Position Design, have than the effect of the decorrelation of wide spectrum virus (containing this viral different subtype) after in importing plant materials.

Term

As used herein, described " plant (or crop) " comprises the plant of any kind, as long as this plant is applicable to carrying out the conversion operation (transgeneic procedure) of gene, as various farm crop, flower plant or forestry plant etc.Described plant is such as being: dicotyledons, monocotyledons or gymnosperm.Such as but not limited to: Chinese cabbage, Plantula Brassicae chinensis that the Cruciferae rape belongs to, Cruciferae mouse ear mustard, paddy rice gramineous, comprise tobacco, melon and fruit, vegetables, rape etc. in addition.More specifically, described plant includes, but is not limited to: wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olea, Sunflower Receptacle, coconut, the Viscotrol C plant, cocoa beans, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, natsudaidai, spinach, the piemarker lettuce, asparagus, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, onion, potato, tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, nut, coffee, eggplant, sugarcane, tealeaves, pepper, grapevine, oyster fiber crops grass, banana, natural rubber tree and ornamental plant etc.

As used herein, unless otherwise indicated, described " virus " is to infect the virus of plant, and its genome comprises the nucleotide sequence of RNA silencing suppressor.For example, described virus is: Brassica 2 et 4 (TuMV) or cucumber mosaic virus (CMV).

As used herein, term " RNA disturbs (RNA interference, RNAi) " refers to that some double-stranded RNAs can block the expression of specific gene in body efficiently, specifically, impels the mRNA degraded, lure that cell shows the phenotype of specific gene disappearance into, it is intervened also referred to as RNA or interferes.

As used herein, described " Microrna (miRNA) " refers to a kind of RNA molecule, is generally 20-23nt.Described miRNA can specific binding to the corresponding site of the mRNA of virogene silencing suppressors, affect the Transcription of mRNA, thereby reach the effect that suppresses viral.

As used herein, unless otherwise indicated, described " oligonucleotide " refers to a kind of DNA sequence dna, and it is corresponding to described miRNA.Described oligonucleotide, after importing vegetable cell, tissue or organ, can be expressed its corresponding miRNA.

As used herein, " basically complementary " refers to that the sequence of Nucleotide is enough complementary, can interact in a kind of foreseeable mode, as formed secondary structure (as loop-stem structure).It is complementary that the nucleotide sequence of two usually, " basically complementary " has 70% Nucleotide between mutually at least; Preferably, it is complementary having 80% Nucleotide at least; Preferred, it is complementary having 90% Nucleotide at least; Further preferred, it is complementary having 95% Nucleotide at least; As 98%, 99% or 100%.Usually, two enough can have maximum 7 unmatched Nucleotide between complementary molecule; Preferably, there are maximum 6 unmatched Nucleotide; Preferred, there are maximum 5 unmatched Nucleotide; Further preferred, there are maximum 4 unmatched Nucleotide, as there is 0,1,2,3,4 unmatched Nucleotide.As used herein, the sequence of " complementation " typically refers to the sequence (as 5 ' ATCG 3 ' → GCTA) that the sequence of 5 '-3 ' direction is converted to its 3 '-5 ' direction, and then gets its complementary sequence (as GCTA → 5 ' CGAT3 ').

As used herein, " stem ring " structure also is known as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form a kind of secondary structure that comprises double-stranded region (stem), described double-stranded region is formed by two zones (being positioned on same a part) of this nucleic acid molecule, the both sides of two double-stranded parts of regional apportion; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. the strand zone.Even two zones of this nucleic acid molecule are not complete complementaries, the double-stranded part of Nucleotide also can keep double-stranded state.For example, insertion, disappearance, replacement etc. can cause not complementary or this zonule self formation loop-stem structure of a zonule or the secondary structure of other form, yet these two zones still can be basically complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, and after the nucleic acid that has obtained a nucleotide sequence with primary structure, those skilled in the art can determine whether this nucleic acid can form loop-stem structure usually.

As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... form ", " basically by ... form " and " by ... form "; " mainly by ... form ", " basically by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

Ultimate principle

In the present invention, the ultimate principle that described RNA disturbs is: using plant as medium, be directed to the miRNA (preferably for high abundance ground, expressing described miRNA) of virogene by plant interior expression by transgenic method, when the virus infection plant and after entering vegetable cell, it can touch the miRNA existed in vegetable cell, described miRNA can be on the mRNA level expression of viral interference gene (as the RNA silencing suppressor gene), thereby suppress copying of virus.

Artificial mi RNA

The inventor carries out sequence alignment by the nucleotide sequence to the RNA silencing suppressor that derives from virus (comprising Brassica 2 et 4 (TuMV) or cucumber mosaic virus (CMV)) different subtype respectively, find out the position of conservative property, based on these position designs, obtained oligonucleotide of the present invention.As shown in the sequence of described oligonucleotide is as arbitrary as SEQ ID NO:1-SEQ ID NO:4.

In addition, the present invention also provides the oligonucleotide of the sequence hybridization that can limit with SEQ ID NO:1-SEQ ID NO:4 under stringent condition, and at least a portion sequence complementation of its corresponding miRNA and the interior RNA silencing suppressor mRNA of virus; Perhaps, shown in arbitrary with SEQ ID NO:1-SEQ ID NO:4, sequence has the oligonucleotide of 95% above homology, and at least a portion sequence complementation of RNA silencing suppressor mRNA in its corresponding miRNA and virus.

According to oligonucleotide sequence provided by the present invention, can be processed into by plant the polynucleotide construction of the miRNA that can affect corresponding mrna expression after can designing in being imported into plant.Therefore, the invention provides construction a kind of separation or artificial constructed, described construction can be transcribed into the miRNA precursor by vegetable cell, and described miRNA precursor can be processed to described miRNA in plant materials.Designing described construction according to oligonucleotide provided by the invention is that those skilled in the art are apprehensible, usually can make this construction comprise an intron sequences (not complementary with the both sides sequence), two ends connect upper complementary gene order, after transfered cell, can produce " neck-ring " structure, and " neck " shape part can be processed the miRNA into about 21-23nt by plant in plant materials, and this miRNA can especially effectively suppress the expression of goal gene.The base of RNA (A, U, C, G) is known in the art with corresponding form or the complimentary fashion of the base (A, T, C, G) of DNA.

As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown at least one formula I:

Seq _forward-X-Seq _oppositelyformula I,

In formula I,

Seq _forwardfor being selected from the oligonucleotide sequence of SEQ ID NO:1-SEQ ID NO:4 shown in arbitrary;

Seq _oppositelyfor with Seq _forwardbasically complementary oligonucleotide sequence;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _oppositelynot complementary.

Structure shown in formula I, after proceeding to vegetable cell, forms the secondary structure shown in formula II:

formula II.

Structure shown in formula II further is sheared, is processed to form miRNA in plant materials, thus the effect of performance gene silencing.

Described construction forms the form more than 1 loop-stem structure after can being prepared in importing body, for example, can comprise the loop-stem structure more than 2 or 2, and these loop-stem structures " neck " shape part is (by Seq _forwardand Seq _oppositelyinteract to form) in complementary oligonucleotide sequence can be respectively: the sequence (corresponding to cucumber mosaic virus (CMV)) that is selected from SEQ ID NO:1 or SEQ ID NO:3; With the sequence that is selected from SEQ ID NO:2 or SEQ ID NO:4 (corresponding to Brassica 2 et 4 (TuMV)).Such construction in importing to plant materials after, the transgenic plant of acquisition can be resisted multiple virus, as resisted cucumber mosaic virus and Brassica 2 et 4 simultaneously.

Improve the plant virus resistance ability

The present invention also provides a kind of method that improves the plant virus resistance ability, and described method comprises step: described construction (a) is provided; (b) construction described in (a) is imported in plant, thus express comprising the corresponding miRNA of described oligonucleotide, described miRNA suppresses the expression of virogene, and then improves the plant virus resistance ability.

With the recombinant DNA conversion of plant, can carry out with routine techniques well known to those skilled in the art, specifically depending on floristic difference.For example, can use the methods such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as Ye Panfa, Rice Young Embryo conversion method etc.Can use ordinary method regeneration plant for the vegetable cell, tissue or the organ that transform.

Usually, described construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described oligonucleotide or the sequence complementary with it.Described expression vector also contains promotor, replication orgin and/or marker gene etc. usually.Method well-known to those having ordinary skill in the art can be for building expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described expression vector preferably comprises one or more selected markers, with the phenotypic character of the host cell that is provided for select transforming, as kalamycin, gentamicin, Totomycin, amicillin resistance.

The carrier that comprises above-mentioned suitable gene order and suitable promotor or control sequence, can be for transforming suitable host.In the method for the invention, described host can be any described expression vector described expression vector can be passed to the host of vegetable cell of being suitable for carrying.Preferably, described host is Agrobacterium.

As preferred embodiment a kind of, the described method that construction is imported to plant comprises:

(1) provide the Agrobacterium of carrying expression vector, described expression vector contains described construction;

(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thereby make described construction proceed to vegetable cell or tissue or organ;

(3) select vegetable cell or tissue or the organ that proceeds to described construction;

(4) vegetable cell in step (3) or tissue or neomorph are become to plant.

The present invention also provides a kind of plant, the corresponding miRNA of the described oligonucleotide of its expression in vivo; Or comprise described polynucleotide construction in its body.Described plant is prepared by aforesaid transgenic method.

Major advantage of the present invention is:

(1) the present invention is applied to the RNA perturbation technique on the transgenosis antivirus plant dexterously, and the transgenosis antivirus plant of development of new, be significant to agriculture development.

(2) sequence by the virogene silencing suppressors to different virus hypotype source compares, and has found the target site that is suitable for carrying out the miRNA interference.Thereby the antiviral plant of the transgenosis prepared by method of the present invention can be resisted the infecting of virus of different virus hypotype.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

I. test materials and method

Material

Arabidopis thaliana Col and Chinese cabbage 99Bre seed: available from Shanghai Agricultural Science and Technology Seeds Co., Ltd..

1. the total RNA of plant tissue extracts

Reagent: TaKaRa RNAiso Reagent extraction agent box.

Step:

A) material is fully ground in liquid nitrogen, add in sample with the amount of 100mg material 1ml extraction buffer, fully mix standing 10 minutes of room temperature.

B) 13000rpm is centrifugal 5 minutes, and supernatant is proceeded in new centrifuge tube, adds 200 μ l chloroforms, fully mixes, and room temperature makes its layering in standing 10 minutes.

C) 13000rpm is centrifugal 5 minutes, carefully draws supernatant in new centrifuge tube.

D) add the equal-volume Virahol, mix rear room temperature standing 10 minutes.

E) 13000rpm is centrifugal 5 minutes, with 1ml 75% (v/v) ethanol, washes once after abandoning supernatant.

F) 7800rpm is centrifugal 5 minutes, abandons low-speed centrifugal after supernatant, with the rifle head, sucks residual liquid, and room temperature is dried, and until RNA, just after drying, adds the appropriate water without RNase, and 65 ℃ are stored in-70 ℃ after within 10 minutes, fully dissolving.

2. the primer of artificial mi RNA expression vector establishment

CMV-2b1 (with respect to 21-41 position in SEQ ID NO:36)

The DNA sequence dna that artificial mi RNA is corresponding: 5 ' TGGAGTTCGACGTTTGTCATT 3 ' (SEQ ID NO:1).

The primer of design:

I?miR-s 5’gaTGGAGTTCGACGTTTGTCATTtctctcttttgtattcc?3’(SEQ?IDNO：5)；

II?miR-a 5’gaAATGACAAACGTCGAACTCCAtcaaagagaatcaatga?3’(SEQ?ID?NO：6)；

III?miR*s 5’gaAACGACAAACGTCCAACTCCTtcacaggtcgtgatatg?3’(SEQID?NO：7)；

IV?miR*a 5’gaAGGAGTTGGACGTTTGTCGTTtctacatatatattcct?3’(SEQID?NO：8)。

TuMV-Hc Pro1 (643-663 position in SEQ ID NO:35)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TCTCTCACCCCATATGAAATT 3 ' (SEQ ID NO:2).

The primer of design:

I?miR-s 5’gaTCTCTCACCCCATATGAAATTtctctcttttgtattcc?3’(SEQ?IDNO：9)；

II?miR-a 5’gaAATTTCATATGGGGTGAGAGAtcaaagagaatcaatga?3’(SEQID?NO：10)；

III?miR*s 5’gaAACTTCATATGGGCTGAGAGTtcacaggtcgtgatatg?3’(SEQID?NO：11)；

IV?miR*a 5’gaACTCTCAGCCCATATGAAGTTtctacatatatattcct?3’(SEQ?IDNO：12)。

CMV-2b2 (110-130 position in SEQ ID NO:36)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TCTCGCTGGGACTTTTGTAAC 3 ' (SEQ ID NO:3).

I?miR-s：5’gaTCTCGCTGGGACTTTTGTAACtctctcttttgtattcc?3’(SEQID?NO：13)；

II miR-a：5’gaGTTACAAAAGTCCCAGCGAGAtcaaagagaatcaatga 3’(SEQ?ID?NO：14)；

III miR*s：5’gaGTCACAAAAGTCCGAGCGAGTtcacaggtcgtgatatg 3’(SEQ?ID?NO：15)；

IV?miR*a：5’gaACTCGCTCGGACTTTTGTGACtctacatatatattcct?3’(SEQID?NO：16)。

TuMV-Hc Pro2 (602-622 position in SEQ ID NO:35)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TGTCGCACATTAGTGTTGGGT 3 ' (SEQ ID NO:4).

I?miR-s：5’gaTGTCGCACATTAGTGTTGGGTtctctcttttgtattcc?3’(SEQID?NO：17)；

II miR-a：5’gaACCCAACACTAATGTGCGACAtcaaagagaatcaatga 3’(SEQ?ID?NO：18)；

III?miR*s：5’gaACACAACACTAATCTGCGACTtcacaggtcgtgatatg?3’(SEQID?NO：19)；

IV?miR*a：5’gaAGTCGCAGATTAGTGTTGTGTtctacatatatattcct?3’(SEQID?NO：20)。

Primer corresponding to pRS300

Primer A:5 ' CTG CAA GGC GAT TAA GTT GGG TAA C 3 ' (SEQ ID NO:21);

Primer B:5 ' GCG GAT AAC AAT TTC ACA CAG GAA ACA G 3 ' (SEQ IDNO:22).

3. the primer of sxemiquantitative RT-PCR

CMV-2b1 reverse transcription primer:

5’＞GTCGTATCCAGTGCGGGTCCGAGGTATTCGCACTGGATACGACaatgaca＜3’(SEQ?ID?NO：23)。

CMV-2b2 reverse transcription primer:

5’＞GTCGTATCCAGTGCGGGTCCGAGGTATTCGCACTGGATACGACgttacaa＜3’(SEQ?ID?NO：24)。

TuMV-Hc Pro1 reverse transcription primer:

5’＞GTCGTATCCAGTGCGGGTCCGAGGTATTCGCACTGGATACGACaatttca＜3’(SEQ?ID?NO：25)。

TuMV-Hc Pro2 reverse transcription primer:

5’＞GTCGTATCCAGTGCGGGTCCGAGGTATTCGCACTGGATACGACacccaac＜3’(SEQ?ID?NO：26)。

The CMV-2b1RT-PCR primer:

Forward: 5 '>TGGAGTTCGACGTTTGTCAT<3 ' (SEQ ID NO:27);

Oppositely: 5 '>TCGTATCCAGTGCGGGTCCG<3 ' (SEQ ID NO:28).

The CMV-2b2RT-PCR primer:

Forward: 5 '>TCTCGCTGGGACTTTTGTAA<3 ' (SEQ ID NO:29);

Oppositely: 5 '>TCGTATCCAGTGCGGGTCCG<3 ' (SEQ ID NO:30).

TuMV-Hc Pro1RT-PCR primer:

Forward: 5 '>TCTCTCACCCCATATGAAAT<3 ' (SEQ ID NO:31);

Oppositely: 5 '>TCGTATCCAGTGCGGGTCCG<3 ' (SEQ ID NO:32).

TuMV-Hc Pro2RT-PCR primer:

Forward: 5 '>TGTCGCACATTAGTGTTGGG<3 ' (SEQ ID NO:33);

Oppositely: 5 '>TCGTATCCAGTGCGGGTCCG<3 ' (SEQ ID NO:34).

Reagent:

AMV ThermoScript II (TAKARA);

RNase inhibitor (TAKARA);

DNase?I(RNase?free)(TAKARA)。

Step:

A) extract respectively total RNA of plant (Arabidopis thaliana or Chinese cabbage) blade, with DNase I (RNase free), process phenol chloroform extracting afterwards in 30 minutes, precipitation, dry up, without the water dissolution of RNase.

B) survey OD260 and electrophoresis quantitatively after, get the total RNA of 1 μ g, the by specification operation, 42 ℃ of reactions 1 hour, 94 ℃ 5 minutes so that the ThermoScript II inactivation.

C) one times of dilution is respectively got 1 μ l after reverse transcription product and is PCR.PCR reaction conditions: 94 ℃ of 3min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 30sec, 25-28 circulation; 72 ℃ of 5min.For proofreading and correct the template amount for the RT-PCR reaction, the primer of Ubiquitin and Actin of usining carries out parallel PCR reaction as internal reference.

4.CTAB the extraction of the total DNA of method plant tissue

Reagent:

2 * CTAB damping fluid (100ml): 10ml 1M Tris pH 8.0; 4ml 0.5M EDTA pH8.0; 8.19g NaCl; 2g CTAB; 1g PVP K30 is settled to 100ml.

1 * CTAB damping fluid (100ml): 5ml1M Tris pH 8.0; 2ml 0.5M EDTA pH8.0; 1g CTAB is settled to 100ml.

High salt TE (100ml): 1ml 1M Tris pH 8.0; 200 μ l 0.5M EDTA pH8.0; 5.844gNaCl be settled to 100ml.

10% (w/v) CTAB (50ml): 5g CTAB; 2.045g NaCl is settled to 50ml.

Step:

A) get in 5g vegetable material liquid nitrogen and proceed in the 40ml centrifuge tube after abrasive flour.

B) add 2 * CTAB damping fluid (1: 1) of 65 ℃ of preheatings of 15ml, in 65 ℃ of incubation 10min, constantly turn upside down several times therebetween after mixing up and down.

C) add the chloroform of 1 times of volume: primary isoamyl alcohol (24: 1) mixes the centrifugal 5min of rear 11000rpm.

D) draw supernatant to new centrifuge tube, fill into 1/10 volume 10%CTAB, then add the chloroform of 1 times of volume: primary isoamyl alcohol mixes rear centrifugal 5min.

E) get supernatant, repeat d) step 2-3 time, supernatant is proceeded to new centrifuge tube after centrifugal for the last time, add the precipitation buffering liquid (1 * CTAB) that is greater than 2 times of volumes, mixing of gentleness placed 30min under room temperature.

F) centrifugal and collecting precipitation, be resuspended in precipitation in the high salt TE of 5ml of 65 ℃, now can add some Rnase, 37 ℃ of incubation 30min.

G) after the centrifugal 10min of 11000rpm, supernatant is transferred in new 1.5ml centrifuge tube.

H) add 2 times of volume dehydrated alcohols, in-20 ℃ of placement 30min, centrifugal after mixing, abandon supernatant, with drying after 70% washing with alcohol, be dissolved in 100 μ l TE.

5. the preparation of freeze-thaw method Agrobacterium competent cell and conversion

A) cultivate the mono-bacterium colony of picking GV3101 the fresh flat board of 48 hours from 28 ℃, forward in 20ml LB liquid nutrient medium (rif 50mg/l, GM 5050mg/l), in 28 ℃ of 250rpm shaking culture spend the night (can not be too dense).(following all operations were all carries out under aseptic condition).

B) ice bath is after 20 minutes, by the centrifuge tube of bacterium liquid packing 5ml (every pipe 4ml), and ice bath 10 minutes.

C) 4000rpm (5-10 ℃) is centrifugal 10 minutes, abandons supernatant liquor.

D) every pipe adds the 20mM CaCl of the abundant precooling of 1ml ₂with resuspended thalline.Ice bath 10 minutes.

E) 4000rpm (5-10 ℃) is centrifugal 10 minutes, abandons supernatant.

F) every pipe adds 300 μ l 20mM CaCl ₂(depending on cell concentration), be in charge of after merging in the centrifuge tube of 1.5ml.

G) every pipe adds 1 μ l plasmid, ice bath 5 minutes, then put into liquid nitrogen 4-5 minute.

H) put 5 minutes for 37 ℃, every pipe adds 400 μ l LB cultivations within 2 hours, to make bacteria resuscitation based on 28 ℃ of incubations, and expresses corresponding antibiotics resistance gene.

I) respectively get 200 μ l volume coated plates, room temperature is placed a little while, in 28 ℃ of cultivations.

6. flower-dipping method arabidopsis thaliana transformation and screening

Reagent:

Transform damping fluid (1L): macroelement (50 *): 10ml; Trace element (1000 *): 0.5ml; CaCl ₂(100 *): 5ml; Molysite (200 *): 2.5ml; Organic (100 *): 10ml; Sucrose: 50g; 6-BA (1mg/ml): 10 μ l; Silwet L-77:400 μ l (vacuum filtration is used 200 μ l); Be adjusted to pH5.8 with KOH, constant volume 1L.

The screening culture medium flat board: 3% (w/v) sucrose MS0 solid medium (pH5.8) adds kantlex (Kan) to 50mg/l.

Step:

A) after the Arabidopis thaliana bolting, stem is high approximately can be transformed 5 centimetres the time, if plant setting percentage to be transformed is low, will after plant is pinched 4 days, carry out.

B) before transforming, flower and the angle of having pollinated are really got rid of.And the soil water suction is spent the night.

C) Agrobacterium of overnight incubation is diluted at 1: 100 in large bottle substratum, cultivate after 24 hours for 28 ℃, centrifugal 20 minutes of 4 ℃ of 5000rpm, abandon supernatant, Agrobacterium is precipitated in the conversion damping fluid of the two volumes that is suspended in original bacteria liquid, made OD600 in 0.8 left and right.

D) over-ground part of Arabidopis thaliana immerses in bacterium liquid 30 seconds fully, and taking-up keeps flat, and covers preservative film and newspaper, under dark, spends the night, and moves into that phytotron is normal vertically to be cultivated next day.After sowing dry 2 weeks.

E) seed is laid in the Ms0 solid plate containing 50mg/l Kan after aseptic sterilization, through 4 ℃ of vernalization, moves on to two days later group training chamber, blocks that resistance seedling and moves on to continued growth in soil.

F) get blade extraction genomic dna and detect and obtain positive seedling through PCR, then screening obtains genetically modified pure lines through two generations, for further analysis.

7. the high pressure permeability method transforms Chinese cabbage and screening

Reagent:

Transform damping fluid (1L): macroelement (50 *): 10ml; Trace element (1000 *): 0.5ml; CaCl ₂(100 *): 5ml; Molysite (200 *): 2.5ml; Organic (100 *): 10ml; Sucrose: 50g; 6-BA (1mg/ml): 10 μ l; Silwet L-77: 200 μ l for vacuum filtration; Be adjusted to pH5.8 with KOH, constant volume 1L.

The screening culture medium flat board: 3% sucrose MS0 solid medium (pH5.8) adds kantlex (Kan) to 50mg/l.

Step:

A) after Chinese cabbage plant bolting, just can be transformed.

B) before transforming, flower and the angle of having pollinated are really got rid of, and the soil water suction is spent the night.

C) Agrobacterium of overnight incubation is diluted at 1: 100 in large bottle substratum, cultivate after 24 hours for 28 ℃, centrifugal 20 minutes of 4 ℃ of 5000rpm, abandon supernatant, Agrobacterium is precipitated in the conversion damping fluid of the two volumes that is suspended in original bacteria liquid, made OD600 in 0.8 left and right.

D) over-ground part of Chinese cabbage immerses in bacterium liquid fully, vacuum infiltration 5 minutes, and intermittently 2 minutes, vacuum infiltration was 5 minutes again, then took out and kept flat, and covered preservative film and newspaper, under dark, spent the night, and moved into normal vertical cultivation of phytotron next day.After sowing dry 2 weeks.

E) seed is laid in the Ms0 solid plate containing 50mg/l Kan after aseptic sterilization, through 4 ℃ of vernalization, moves on to group training chamber after 30 days, blocks that resistance seedling and moves on to continued growth in soil.

F) get blade extraction genomic dna and detect and obtain positive seedling through PCR, then screening obtains genetically modified pure lines through two generations, for further analysis.

8. plant virus inoculation method (artificial friction inoculation)

1) first rinse well with tap water on the test plant blade face, sprinkles equably friction agent (silicon carbide or diatomite) after drying.

2) blade of clip fritter virus disease sample is placed in mortar, adds a small amount of phosphate buffered saline buffer, grinds into pasty state.

3) dip a small amount of sick leaf sap with forefinger or pestle, gently smear 2-3 time at the plant leaf surface for inoculation, notice that friction is light.Do not wipe back and forth, answer uniaxially to smear to the blade tip direction from phyllopodium, the inoculation leaf is held with palm or finger, firmly not excessive, more can not stave blade.

4) after the inoculation, be that sick juice and the friction agent on blade face are rinsed in available wash bottle well.Attention: friction agent must be rinsed out, otherwise will affect observation of symptoms in the future.

5) sting the mark of an aperture as the inoculation leaf with pencil in the point of blade of inoculating.

6) each group will be made blank with a basin plant, with damping fluid, replaces the disease leaf sap to do inoculation.

7) finish writing after label and insert in flowerpot with pencil.

8) plant of taking over kind will be placed in the greenhouse of illumination and cultivate, and observes the record incidence.

9) symptom record: susceptible phenotype chlorisis occurs, withered spot and necrosis for inoculating blade and young leaves; Disease-resistant phenotype chlorisis do not occur, withered spot and necrosis for inoculating blade and young leaves.

II. embodiment

The Fine design of embodiment 1, antiviral miRNA

The inventor has collected 56 Brassica 2 et 4 TuMV genome sequences that source is different, full length sequence to its silencing suppressors HC-Pro is analyzed, also collected 11 cucumber mosaic virus CMV genome sequences that source is different simultaneously, the full length sequence of its RNA silencing suppressor 2b has been analyzed.Sequence contrast partial results is shown in Fig. 3 and Fig. 4.By the sequence alignment analysis, select respectively small segment that conservative property is strong as possible candidate's artificial mi RNA target sequence from RNA silencing suppressor HC-Pro and 2b sequence.

In order more effectively to design the artificial mi RNA plant expression vector, the 2b full length sequence (SEQ ID NO:36) of HC-Pro full length sequence based on TuMV (SEQ ID NO:35) and CMV, the inventor analyzes the secondary structure of these two RNA silencing suppressors, sees respectively Fig. 5 and Fig. 6.On above-mentioned homology contrast basis, for the sequence area that there is no special secondary structure, further screen, select the strong zone that does not produce special secondary structure of conservative property as the artificial mi RNA target position simultaneously, effectively design the artificial mi RNA expression vector.

Through a large amount of comparison and test, result obtains the DNA sequence dna corresponding with artificial microRNA nucleotide sequence shown in following SEQ ID NO:1-4.Although SEQ ID NO:1-4 designs according to corresponding section in SEQ ID NO:35 and SEQ ID NO:36, but there is the adjustment in indivedual bases site with respect to corresponding site in SEQ ID NO:35 and SEQ IDNO:36, to be reference source adjusted in the HC-Pro of most TuMV or CMV virus or 2b sequence homology comparing result for this, for majority source conserved sequence, designs artificial miRNA sequence.

CMV-2b1 (with respect to 21-41 position in SEQ ID NO:36)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TGGAGTTCGACGTTTGTCATT 3 ' (SEQ ID NO:1).

Corresponding artificial microRNA nucleotides sequence is classified as: 5 ' UGGAGUUCGACGUUUGUCAUU3 ' (SEQ ID NO:37).

TuMV-Hc Pro1 (643-663 position in SEQ ID NO:35)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TCTCTCACCCCATATGAAATT 3 ' (SEQ ID NO:2).

Corresponding artificial microRNA nucleotides sequence is classified as: 5 ' UCUCUCACCCCAUAUGAAAUU3 ' (SEQ ID NO:38).

CMV-2b2 (110-130 position in SEQ ID NO:36)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TCTCGCTGGGACTTTTGTAAC 3 ' (SEQ ID NO:3).

Corresponding artificial microRNA nucleotides sequence is classified as: 5 ' UCUCGCUGGGACUUUUGUAAC3 ' (SEQ ID NO:39).

TuMV-Hc Pro2 (602-622 position in SEQ ID NO:35)

The corresponding DNA sequence dna of artificial mi RNA: 5 ' TGTCGCACATTAGTGTTGGGT 3 ' (SEQ ID NO:4).

Corresponding artificial microRNA nucleotides sequence is classified as: 5 ' UGUCGCACAUUAGUGUUGGGU3 ' (SEQ ID NO:40).

Embodiment 2, artificial mi RNA plant expression vector construction

With reference to The Plant Cell (2006), Vol.18, the method provided in 1121-1133.

The artificial mi RNA expression vector establishment utilizes carrier pRS300 (available from the general developmental biology of German horse institute, carrier carries pre-miR319a) for template, and Fig. 1 is shown in by its collection of illustrative plates.

As shown in Figure 2, main route is to replace the ripe small segment of miR319 in the pre-miR319a that is present in carrier pRS300 with artificial mi RNA in the mode of rite-directed mutagenesis to cloning process, builds the plant gene expression vector of antiviral artificial mi RNA:

1) do you each sequence in aforementioned " the corresponding DNA sequence dna of artificial mi RNA nucleotide sequence " is input to respectively to Net-based Design instrument WMD-Web MicroRNA Designer (http://wmd.weigelworld.org/cgi-bin/mirnatools.pl? page=4) in, produce the artificial microRNA expression vector establishment of following structure combination of primers, primer I: microRNA forward (miR-s), II:microRNA reverse (miR-a), III:microRNA*forward (miR*s), IV:microRNA*reverse (miR*a), take turns doing PCR (a) in conjunction with primer A and B according to the combination of primers of coupling shown in table 1, (b), (c) and (d).For PCR (a), (b), (c) reaction conditions is at first 94 ℃, and then 2 minutes be 94 ℃ of 30 circulations, 15 seconds, 60 ℃, 30 seconds, 68 ℃, 50 seconds, uses the KOD plus enzyme (Takara, Dalian) of high-fidelity.Be at first 94 ℃ for PCR (d) reaction conditions, then 2 minutes be 94 ℃ of 30 circulations, 15 seconds, 60 ℃, 30 seconds, 68 ℃, 1 minute 40 seconds, uses the KOD plus enzyme (Takara, Dalian) of high-fidelity.

Table 1. artificial mi RNA vector construction PCR combination of primers

	Forward primer	Reverse primer	Template
				(a)	A	IV	pRS300
(b)	III	II	pRS300
				(c)	I	B	pRS300
(d)	A	B	(a) PCR product+(b)+(c)

2) PCR (d) fragment has comprised PCR (a), and (b) and (c) fragment, then with Kpn I and Xba I double digestion PCR (d) fragment and carrier pRS300 respectively.

3) the PCR fragment of double digestion and carrier are connected to screening positive clone with the T4DNA ligase enzyme.Result, obtain 5 and cross the expression plant vectors, respectively containing just like construction amiR-2b1, the amiR-2b2 shown in Fig. 2 right side, amiR-HC Pro1, amiR-HC Pro2 and amiR-2b1/HC Pro1 (construction amiR-2b1 and amiR-HC Pro1 are connected on end to end in a carrier and express).

4) utilize freeze-thaw method that the artificial mi RNA carrier of structure is proceeded in GV3101 Agrobacterium (purchased from Invitrogen).

Embodiment 3, Arabidopis thaliana transfer-gen plant phenotype

By homologous sequence, contrast and secondary structure analysis, the inventor has selected respectively 4 small segments in RNA silencing suppressor HC-Pro and 2b sequence as the artificial mi RNA target position, build 5 and crossed the expression plant vector, contain respectively the amiR-2b1/HC Pro1 of construction amiR-2b1, amiR-2b2, amiR-HC Pro1, amiR-HC Pro2 and joint mapping thing amiR-2b1 and amiR-HC Pro1, and be transformed in Arabidopis thaliana and Chinese cabbage.In order to identify the artificial mi RNA function, after in transfer-gen plant T2 generation and T3 generation, screened, choose positive transfer-gen plant and carry out the disease resistance check, be that virus inoculation (CMV and TuMV virus strain are from Zhejiang University plant virus laboratory) is to plant, observe plant for viral resistivity, the results are shown in Table 2 and table 3.With wild-type Arabidopis thaliana (WT-Col) in contrast.

Table 2. Arabidopis thaliana transfer-gen plant viral diseases

Wherein, the account form of disease resistance is: (inoculation plant number-disease plant number)/inoculation plant number.

Table 3. Arabidopis thaliana transfer-gen plant viral diseases

Embodiment 4, Chinese cabbage transfer-gen plant phenotype

The inventor utilizes the high pressure permeability method to cross the expression plant vector by 5, is respectively amiR-2b1, amiR-2b2, amiR-HC Pro1, amiR-HC Pro2 and an amiR-2b1/HC Pro1 who combines 2b and HC Pro, is transformed in Chinese cabbage.Identify the artificial mi RNA function, after transfer-gen plant T2 generation and T3 generation are screened, choose positive transfer-gen plant and carry out antiviral (CMV and TuMV are from Zhejiang University plant virus laboratory) property check, in Table 4 and table 5.With wild-type Chinese cabbage (WT-Bre) in contrast.

Table 4. Chinese cabbage transfer-gen plant viral diseases phenotype

Table 5. Chinese cabbage transfer-gen plant viral diseases phenotype

Embodiment 5, detection artificial mi RNA are expressed in the Arabidopis thaliana transfer-gen plant

Utilize RT-PCR, adopt stem ring reverse transcription primer pair miRNA to carry out special RT-PCR, detect miRNA at plant interior expression.Test principle is as Fig. 7.

Utilize stem ring reverse transcription primer (as listed reverse transcription primer in afore-mentioned test materials and methods " the 3. primer of sxemiquantitative RT-PCR "), the miRNA reverse transcription of expression can be become to cDNA; Take this cDNA as template, carry out RT-PCR in conjunction with forward and reverse primer (being listed RT-PCR primer in afore-mentioned test materials and methods " the 3. primer of sxemiquantitative RT-PCR "), amplified production is checked with agarose gel electrophoresis, result is as Fig. 8, in the transfer-gen plant of the resistant panel positive, miRNA obtains expression.

Conclusion

The above results shows, artificial mi RNA can effectively be controlled Chinese cabbage virus disease, can be for improvement of Chinese cabbage group viral diseases ability.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (9)

1. a separation or artificial constructed oligonucleotide, described oligonucleotide is selected from:

(i) SEQ ID NO:1-SEQ ID NO:4 arbitrary shown in the oligonucleotide of sequence; Or

(ii) with the oligonucleotide of the arbitrary sequence complementation (i) limited.

2. a construction, described construction contains oligonucleotide claimed in claim 1, and it can express the miRNA of (i) described oligonucleotide in claim 1 after importing vegetable cell, tissue or organ;

Described construction contains at least one group of structure shown in formula I:

Seq _forward-X-Seq _oppositelyformula I;

In formula I,

Seq _forwardfor oligonucleotide sequence claimed in claim 1;

Seq _oppositelyfor with Seq _forwardcomplementary oligonucleotide sequence;

X is for being positioned at Seq _forwardand Seq _oppositelybetween intervening sequence, and described intervening sequence and Seq _forwardand Seq _{instead to}not complementary.

3. construction as claimed in claim 2, is characterized in that, in described construction, contains:

Be selected from the sequence of SEQ ID NO:1 or SEQ ID NO:3; With

Be selected from the sequence of SEQ ID NO:2 or SEQ ID NO:4.

4. construction as claimed in claim 2, is characterized in that, described construction is expression vector.

5. construction as claimed in claim 2, is characterized in that, the structure shown in formula I, after proceeding to vegetable cell, forms the secondary structure shown in formula II:

formula II,

In formula II, Seq _forward, Seq _oppositelywith stating as defined above of X,

|| be illustrated in Seq _forwardand Seq _oppositelybetween the relation of complementation.

6. the purposes of oligonucleotide claimed in claim 1 or the described construction of claim 2-5 any one, described oligonucleotide orconstruction is for the preparation of antiviral reagent; Or described oligonucleotide orconstruction is for the preparation of the reagent that improves the plant virus resistance ability; Described virus is: Brassica 2 et 4 (Turnip mosaic virus.) or cucumber mosaic virus (Cucumber mosaic virus.).

7. a method that improves the plant virus resistance ability, is characterized in that, described method comprises step:

(a) provide the described arbitrary construction of claim 2-5;

(b) construction described in (a) is imported in plant;

Described virus is: Brassica 2 et 4 (Turnip mosaic virus.) or cucumber mosaic virus (Cucumber mosaic virus.).

8. method as claimed in claim 7, is characterized in that, the described method that construction is imported to plant comprises:

(1) provide the Agrobacterium of carrying the described arbitrary construction of claim 2-5;

(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thereby make described construction proceed to plant.

9. a miRNA, its be SEQ ID NO:37-SEQ ID NO:40 arbitrary shown in the miRNA of nucleotide sequence.

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