CN101429513B - RNA induced silencing complex mediated shearing site and uses thereof - Google Patents

Abstract

The invention discloses a shear locus mediated by RNA induced silencing complex and application thereof. The locus can be applied in designing a factitial tiny RNA. In a DNA molecule coding a factitial tiny RNA precursor, the factitial tiny RNA precursor is a single-chain RNA capable of generating a tiny RNA in plant; the single-chain RNA forms a stem-loop structure; a nucleotide sequence containing sequence 1 in a sequence list is arranged in a fragment of the stem part of the stem-loop structure; two base pairs which are not complementary are arranged on the stem part of the stem-loop structure; and the two uncomplementary base pairs correspond to the ninth position and the tenth position from tail end of 5' in the sequence 1. The invention also discloses a method for culturing plant resisting cucumber mosaic virus. With the DNA molecule coding the factitial tiny RNA precursor, the plant with high efficient virus resistance can be obtained, so that the method is a biological engineering method with economy and efficiency.

Description

Shearing site that the RNA inductive is silencing complex mediated and application thereof

Technical field

The present invention relates to silencing complex mediated shearing site of RNA inductive and application thereof.

Background technology

The plant RNA silencing system is a kind of conservative mechanism that extensively exists in eukaryote, and its mode of action is to make sequence complementary target RNA degraded with it through the route of synthesis generation microRNA of a series of complexity and with site-specific cut mode.These little RNA length are many at 21-24nt, mainly comprise two kinds of Microrna (microRNA) and siRNAs (siRNA).The strand hairpin structure that short microRNA precursor has, (artificial microRNA amiRNA) can efficiently specially induce the native gene silence to the artificial microRNA similar to the microRNA front body structure in plant.Nearest studies show that, amiRNA also has good application potential as a kind of efficient gene silencing instrument in plant virus resistance research.

The RNA silence of microRNA mediation has been widely used in knock out (Baulcombe, 2004,2005) to target gene.Can directly apply to body outer cell line culture (McMaus and Sharp, 2002 such as human Hela cell and fruit bat S2 cell as siRNA; Tuschl et al., 1999; Zamore et al., 2000).In plant, have and a kind ofly can or have the system that the precursor RNA s of hairpin structure transforms long dsrna s (dsRNAs), in this system, the DCL of RNaseIII type (Dicer like protein) albumen can cut into siRNAs or microRNAs with long dsrna s (dsRNAs) or the precursor RNA s that has a hairpin structure, then little RNA with contain the proteic mixture of Argonaute (AGO) (RNA inductive silencing complex) and combine (Bartel, 2004; Carrington and Ambros, 2003; Kim, 2005; Park et al., 2002; Zamore et al., 2005), RNA identification and its sequence complementary target mRNA, under the guidance of the little RNA of antisense, little RNA and RNA inductive silencing complex bonded mixture are by sequence complementary pairing mechanism identification target RNA, and shear at specific site, shearing site usually occurs between 10-11 the Nucleotide of the little RNA 5 ' end of antisense, the target mRNA that is sheared and then be degraded (Khvorova et al., 2003; Llave et al., 2002a; Schwarzet al., 2003; Vaucheret et al., 2004).

In Arabidopis thaliana and paddy rice, in order to express special microRNAs, (artificial microRNAs is effectively expressing frame (Alvarez et al., 2006 of inducing the specific gene silence for the short hairpin RNA s on basis amiRNAs) with the microRNA precursor; Chen et al., 2004; Guo et al., 2005; Ossowski et al., 2008; Parizotto et al., 2004; Schwab et al., 2006; Wang et al., 2005; Warthmann et al., 2008; ).Recently, in Plant Biotechnology, amiRNA is reported (Qu et al., 2007 successively as the application potential of antiviral method; Niu et al., 2006).

In past 10 years, by express viral source justice, antisense sequences or complementaryly double-strandedly come, the RNA silence of mediation has been widely used in plant virus resistance gene engineering (Helliwell and Waterhouse, 2005; Smithet al., 1994; Waterhouse et al., 1998).In these antiviral engineerings, the RNA silence is the viral RNA generation inhibition altogether by the transgenosis RNA of viral source and intrusion, and then makes plant produce very strong resistance or recover phenomenon (Guo and Garcia, 1997 virus; Linbo et al., 1994).Compare with the antiviral silence of long RNA mediation, amiRNA has many good qualities: at first, do not need long viral cDNA fragment, a weak point do not have the amiRNA sequence of homology can avoid non-specific shearing with plant gene, the phenomenon of promptly " missing the target ".Secondly, the antiviral application in agriculture production of amiRNA mediation can not brought the Biosafety problem, in plant, express amiRNAs, need not consider that the virus sequence in the transgenic plant can be with non-target virus complementary or recombinate (Garciaand Simon-mateo, 2006).

In Arabidopis thaliana, express sub at the silence inhibition of turnip yellow virus (TYMV) and Brassica 2 et 4 (TuMV) design respectively---the amiRNA of P69 and HC-Pro albumen coded sequence, the render transgenic plant has obtained respectively the resistance of TYMV and TuMV (Niu et al., 2006).Yet the silence of expressing at cucumber mosaic virus (CMV) coding suppresses son---the amiRNA transgene tobacco of 2b infects CMV and produces different reactions, comprises resistance, recovery type, postpones (Qu et al., 2007) such as resistance and sensitivities.The reason that resistance is not strong may be that CMV comprises three geneome RNAs and two subgenomic RNAs, CMV geneome RNA 1 and RNA3, and subgenomic RNA 4 is not the direct target of amiR2b, and derive from siRNAs quick copy in vegetable cell of these non-target RNA, these siRNA combine with the RISC mixture, and then cause the saturated of siRNA and RISC complex body, influence the antiviral efficient of amiR2b, also had one may be exactly that the target site of amiR2b is not at the optimum target identified region of siRISC complex body.It has been generally acknowledged that for a certain specific mRNA target, be not all complementary siRNA is same effectively with it, RNA is reticent, and efficient not only depends on siRNA sequence itself, also relevant with the higher structure around the target mRNA target spot, higher structure mainly influences the accesibility of siRNA-RISC mixture.

There are some researches show, the infectivity that has the mosaic plumpox virus (PPV) of endogenous function micro RNAs target spot, insert strong and weak different (the Simon-Mateo and Garcia of difference in site because of micro RNA target spot, 2006), the toxicity that is mosaic PPV also depends on the position of micro RNA target sequence in viral genome, shows that some site more helps the shearing of micro RNA-RISC mediation than other site.In addition, in the plant that the PPV mosaic infects, found the selectivity PPV mutant that opposing micro RNA-RISC shears, these suddenly change, and some occurs in the target sequence of micro RNA, the main mutual identification that influences micro RNA5 ' terminal nucleotide and target mRNA with combine, show virus a kind of mechanism of can evolving out, micro RNA shear active negative pressure under escape the RNA silence (Simon-Mateo and Garcia, 2006) of microRNA-RISC mediation.

In zooblast, can utilize influence (Ameres et al., 2007 of the accesibility of biological chemistry and information biology means analysis target spot to siRNA-RISC complex body catalytic activity; Brown et al., 2005; Shao et al., 2007).Though can carry out the computer simulation prediction and estimate of the influence of this structure the target RNA higher structure to the target spot accesibility by the information biology means, but, owing to lack the method that accurately and stably to predict long-chain RNA higher structure, therefore be difficult to the true higher structure of prediction viral RNA in the natural infection process, because can not only there be single rock steady structure in viral RNA, this has just brought very big obstacle for the further application of RNA silence in medical science and agriculture production of siRNA mediation.

CMV is typical triad strand justice RNA viruses (Sugiyama, 2000), comprises three subgroup: IA, IB and II (Roossinck, 1999).CMV nucleic acid is by 3 genomes (RNA1, RNA2 and RNA3) and 2 subgenomic RNAs (RNA4A and RNA4, transcribe by RNA2 and RNA3 respectively) form, as shown in Figure 1, wherein (3 ' UTR) nucleotide sequence height is similar for different RNA 3 ' non-translational region, and 3 ' end of 3 ' UTR can both form higher structure-TLS structure (tRNA like structure) (Kwon and Chung, 2000 that are similar to tRNA; Rietveld et al., 1983).Studies show that, the TLS zone exists the synthetic needed promotor of CMV strand RNA, RNA polymerase (the RdRP that can rely on the RNA of encoding viral, 1a and 2a albumen as CMV RNA1 and RNA2 coding) interact, therefore in the reproduction process of viral RNA, play an important role, and then influence infect (Fernandez-Cuartero et al., 1994) of CMV.The importance of duplicating for CMV in view of CMV 3 ' UTR and the conservative property of 3 ' UTR nucleotide sequence and three-dimensional structure, so CMV 3 ' UTR is well suited for the reticent antiviral target area as RNA.

Summary of the invention

The purpose of this invention is to provide silencing complex mediated shearing site of RNA inductive and application thereof.

The present inventor discovers that 2051/2052 site, 3 ' UTR zone that is positioned at SD type cucumber mosaic virus geneome RNA 3 is the silencing complex mediated shearing site of RNA inductive, this site can be used in the artificial Microrna of design, artificial Microrna is the efficient gene silencing instrument, can be used for plant virus resistance.

Wherein, the nucleotides sequence of the artificial Microrna of 2051/2052 site, 3 ' UTR of target cucumber mosaic virus geneome RNA 3 zone design is classified sequence 1 in the sequence table as.The encoding gene of described artificial Microrna is that coding comprises the 2051/2052nd site, the genomic 3 ' UTR of cucumber mosaic virus zone, and with the cucumber mosaic virus genome complementation of these both sides, site, length is the dna molecular of the RNA of 21～24nt.

For specifically expressing in plant goes out artificial Microrna of the present invention (amiRNA), need to make up the dna molecular of the described amiRNA precursor of described coding, described artificial Microrna precursor is the single stranded RNA that can produce Microrna in plant, this single stranded RNA forms loop-stem structure, the nucleotide sequence that contains sequence 1 in the ordered list in the fragment of the stem of described loop-stem structure, the stem of described loop-stem structure has two base pairs not complementary, two base pairs of described not complementary corresponding to described sequence 1 from 5 ' terminal the 9th and the 10th.

Wherein, described plant is tobacco or Arabidopis thaliana.The nucleotide sequence of the dna molecular of described coding amiRNA precursor specifically can be shown in sequence in the sequence table 2.

The recombinant expression vector, transgenic cell line and the reorganization bacterium that contain above-mentioned dna molecular also belong to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of the dna molecular of described coding amiRNA precursor.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries the dna molecular of described coding amiRNA precursor of the present invention can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.

When using the dna molecular structure recombinant plant expression vector of described coding amiRNA precursor, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (gentamicin marker, kantlex marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.

Another object of the present invention provides a kind of method of cultivating the plant of cucumber-mosaic-virus resistant.

The method of the plant of cultivation cucumber-mosaic-virus resistant provided by the present invention is that the dna molecular with described coding amiRNA precursor changes in the plant, obtains the transgenic plant of cucumber-mosaic-virus resistant.

Described plant can be dicotyledons or monocotyledons, specifically can be Arabidopis thaliana or tobacco.

The present inventor has found that one of the siRNA-RISC mediation is sheared focus, and with this point of contact for target spot designs artificial Microrna, made up the plant recombination expression vector of the dna molecular that contains described coding amiRNA precursor, this carrier is imported in Arabidopis thaliana and the tobacco, change the Arabidopis thaliana and the tobacco of the dna molecular of described coding amiRNA precursor, all show the height resistance that CMV is infected, and can resist not that homophyletic is infecting of CMV.Utilizing the dna molecular of coding amiRNA precursor of the present invention, thereby obtain the plant of efficient virus resistance, is a kind of economical and effective biological engineering method.

Description of drawings

Fig. 1 is the genome structure of CMV

Fig. 2 is that the northern blot hybridization of the transient expression of Q-CMV subgene group clone and the RNA13 ' terminal sequence that contains complete 3 ' UTR and the viral little RNA that 3 ' UTR originates detects

Fig. 3 infects the distribution situation of shearing focus on Q-/SD-/FNY-CMVRNA3 3 ' UTR that the different genotype plant obtains for Q-/SD-CMV and at the amiRNA of isoschizomer design not

Fig. 4 detects for the northern blot hybridization that SD-CMV infects the vsiRNA in wild-type Arabidopis thaliana and dcl2/3/4 mutation type surface analysis (A) and 3 ' UTR source

Fig. 5 is the artificial mi RNA precursor based on Arabidopis thaliana miR159a precursor backbone

Fig. 6 is the expression level of AT3G29250

A:amiR-SD-3 and AT3G29250 are formed with the incomplete pairing of 3 mispairing;

B: sxemiquantitative RT-PCR experiment detects the expression level of AT3G29250.

Fig. 7 is the resistance analysis of amiRNA transgenic arabidopsis for SD-CMV

Fig. 8 is the resistance analysis of amiRNA transgene tobacco for CMV

Embodiment

Embodiment 1, target are in the design of the amiRNA in 2051/2052 site, cucumber mosaic virus genomic 3 ' UTR zone

1) cucumber mosaic virus (CMV) 3 ' UTR can be discerned by the reticent approach of plant endogenous RNA

Can 3 ' UTR zone by identification of plant RNA silencing system and target degraded in the CMV infection processs in order to study, in view of the CMV genome oversize, in order to keep the natural pleated sheet structure in 3 ' UTR zone better, made up expression vector 35S-R4A and the 35S-R4 that comprises complete subgenomic RNA 4A of Q-CMV and RNA4.The construction process of expression vector 35S-R4A and 35S-R4 is as follows: with pQCD2 (Ding, S.W., J.P.Rathjen, W.X.Li, R.Swanson, H.Healy, and R.H.Symons.1995.Efficient infectionfrom cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector.J.Gen.Virol.76 (Pt 2): 459-464) (Institute of Microorganism, Academia Sinica) carries out pcr amplification and obtains RNA4 with QR4-5 (5 '-TCTAGAAGCGTTTAGTTGTTCACCTG-3 ') and QR4-3 (5 '-GAGCTCTGGTCTCCTTATGGAGAAC-3 ') primer for template;

With pQCD3 (Ding, S.W., J.P.Rathjen, W.X.Li, R.Swanson, H.Healy, and R.H.Symons.1995.Efficient infection from cDNA clones of cucumbermosaic cucumovirus RNAs in a new plasmid vector.J.Gen.Virol.76 (Pt2): 459-464) (Institute of Microorganism, Academia Sinica) is template, QR4A-5 (5 '-TCTAGAGTTTTGTATATCTGAGTTCC-3 ') and QR4A-3 (5 '-GAGCTCTGGTCTCCTTATGGAGAACC-3 ') for carrying out pcr amplification, primer obtains RNA4A, RNA4 and RNA4A after order-checking is identified again after XbaI-SacI digestion, be cloned into binary vector pCAMBIA-1300221 (Guo, H.S., Q.Xie, J.F.Fei, and N.H.Chua.2005.MicroRNA directs mRNA cleavageof the transcription factor NAC1 to downregulate auxin signals forarabidopsis lateral root development.Plant Cell 17:1376-1386) in (Institute of Microorganism, Academia Sinica), difference called after 35S-R4 and 35S-R4A.

Because RNA1 does not have the subgene group, from comprising the pQCD1 plasmid (Ding of Q-CMV RNA1 full length cDNA sequence, S.W., J.P.Rathjen, W.X.Li, R.Swanson, H.Healy, and R.H.Symons.1995.Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAsin a new plasmid vector.J.Gen.Virol.76 (Pt 2): 459-464) (Institute of Microorganism, Academia Sinica) downcuts the fragment of RNA1 3 ' end 962 nt through Xho I-BamHI, be cloned on the binary vector pCAMBIA-1300221, obtain recombinant plasmid 35S-R1*.The Agrobacterium that will contain 35S-R1*, 35S-R4A and 35S-R4 plasmid is injected this life cigarette (Nicotiana.benthamiana) respectively and carries out transient expression, injection back 1,2,3,4 day (dpi) difference clip injection blade, extract total RNA, the total RNA of 10 μ g carries out conventional northern blot hybridization after the denaturing formaldehyde gel electrophoresis changes film.Hybridization probe is the special sequence of Q-CMVRNA3 3 ' UTR of [α-32P] dCTP mark.The total RNA of 30 μ g carries out siRNAnorthern blot hybridization after film is changeed in the PAGE gel electrophoresis simultaneously, and hybridization probe is the special in-vitro transcription product of Q-CMV RNA3 3 ' UTR through [α-32P] UTP mark.

SiRNA northern blot results of hybridization as shown in Figure 2, show and inject within back 4 days, the accumulation volume of the siRNA in Q-CMV3 ' UTR source increases gradually, and this explanation Q-CMV RNA can be identified as the substrate of RNA silencing system in the plant materials and be processed into the siRNA molecule.In Q-CMV RNA results of hybridization, can detect the accumulation of Q-CMV RNA1*, RNA4A and RNA4 at an easy rate, these RNA reach peak value at 2-3 days accumulation volumes, and the 4th day begins to descend; And, below Q-CMV RNA hybridization signal, can also detect some along with the continuous enhanced hybridization signal of the prolongation of inject time, this illustrates possible 3 ' UTR regional or concentrated shearing of some quilt at some, exist some to shear focus in other words, thereby caused the accumulation of specific fragment, made the accumulation volume of Q-CMV RNA reduce.But this shearing is that caused by Dicer identification 3 ' UTR processing shearing that cause or the siRNA-RISC mixture that the siRNA that 3 ' UTR originates mediates and unclear.

2) CMV 3 ' UTR shears determining of focus

In order to study the source of the specific fragment that obtains in the injection experiment, designed outside primer and inner primer carries out 5 ' RLM-RACE experiment at Q-CMV RNA3 3 ' UTR least significant end, the point of contact of detecting Q-CMV RNA3 3 ' UTR zone distributes.Adopt the First Choice RLM-RACE test kit of Ambion company, 2ug 35S-R4 injects total RNA of the 4th day of this life cigarette and directly is connected with 5 ' RACE RNA Adapter, and concrete operations are with reference to its instruction manual.The PCR fragment that obtains is connected to (Promega company) on the pGEM-T Easy carrier, selects mono-clonal and carries out sequencing analysis.

Sequencing result and Q-CMV RNA3 3 ' UTR carry out sequence alignment and find, in the clone of 9 order-checkings, there are 6 to be positioned between Q-CMV RNA3 (M21464) 2115～2116 Nucleotide, 3 between 2114～2115 Nucleotide, and these two sites all are positioned at higher structure (TLS) zone that can form similar tRNA, as shown in Figure 3.

Whether CMV 3 ' UTR zone also exists this shearing focus under the natural infection condition in order to study, and it is detected whether consistent in shearing focus and injecting transient expression experiment, the total RNA that infects wild-type Nicotiana glutinosa (N.glutinosa) extraction with Q-CMV is that sample carries out 5 ' RACE nest-PCR, sequencing result and Q-CMV RNA33 ' UTR are carried out sequence alignment, the point of contact that obtains marks with light grey filled arrows in Fig. 3, in whole 19 clones, detect 16 different point of contacts, have only 2 to be positioned at the TLS district that can form higher structure in 16 point of contacts, and the point of contact that obtains with injection experiment is inconsistent, other 14 point of contacts all are positioned at 5 outside 3 ' UTR TLS zone ' end, and this zone can not form very high-grade structure.Further analysis can be found, all between Q-CMV RNA3 2007～2023 Nucleotide, this is the hot spot region (hotspot region) of a shearing to the major part (8) in 14 point of contacts.This experimental result shows that in CMV natural infection process, RNA3 3 ' UTR shears 5 ' end that focus mainly occurs in 3 ' UTR outside the TLS district.

Because Q-CMV is a hypotoxicity strain system in the field, in order to make the shearing focus can be applied to antiviral research better, having chosen the strong virus force strain again is SD-CMV (subgroup IB), equally at outside primer of RNA3 3 ' UTR 3 ' tip designs and inner primer, total RNA that selection is extracted from the wild-type Arabidopis thaliana of SD-CMV natural infection carries out 5 ' RACE nest-PCR, sequencing result and SD-CMV RNA3 3 ' UTR are carried out sequence alignment, the point of contact that obtains is marked among Fig. 3 with the solid black arrow, in whole 31 clones, obtain 27 different point of contacts altogether, and these 27 point of contacts all are positioned at 5 ' end of upstream, TLS zone.Wherein, be distributed with 12 point of contacts between SD-CMV RNA3 2045～2072 Nucleotide, form one and shear the hot spot region, this is close with the shearing hot spot area location that obtains at Q-CMV infection wild-type Nicotiana glutinosa.

Detected point of contact, front is all infected in the wild-type plant at CMV and is obtained, shear the point of contact of causing by DCL albumen in order to eliminate those as far as possible, search out those point of contacts by siRNA-RISC processing, infect Arabidopis thaliana DCL albumen Trimutant with SD-CMV, infect that obvious susceptible symptom appears in system's leaf after 10 days, shown in Fig. 4 A, among Fig. 4 A, " Col-0 " represents the wild-type Arabidopis thaliana, and " dcl2-1/3-1/4-2 " represents Arabidopis thaliana DCL albumen Trimutant; " Mock " represents the Arabidopis thaliana of uninfecting virus, and " SD-CMV " represents the Arabidopis thaliana of SD-CMV virus infection; Mutant is grown severe deformities, comprises plant dwarfing, inflorescence and bending blade, the delay etc. of blooming.In order to detect the accumulation whether SD-CMV infects the little RNA (vsiRNA) in CMV source behind the dcl2-1/3-1/4-2 mutant, synthesized the 21nt oligonucleotide chain in several SD-CMV RNA3 3 ' UTR sources at random, through [γ- ³²P] behind the ATP end mark as probe, extract respectively that SD-CMV infects back 16 days wild-type Col and the total RNA of dcl2-1/3-1/4-2 mutant system infected leaves carries out northern blot hybridization.Results of hybridization is shown in Fig. 4 B, in the wild-type Arabidopis thaliana Col that infects (representing with " WT " among Fig. 4 B), detected the accumulation of a large amount of vsiRNA, and very faint accumulation is arranged also in the dcl2-1/3-1/4-2 mutant, be presented under the situation of the main DCL protein delation in the gene silencing approach of virus induction, possible DCL1 can bring into play part and shear function, it would, of course, also be possible to dcl2-1/3-1/4-2 is not the complete deletion mutant of DCL protein function, or the homologous protein of other class Dicer is processed the point of contact of causing.The small amount of accumulation of vsiRNA provides experimental basis for screening vsiRNA-RISC shearing site.

The total RNA of the susceptible Ye of dcl2-1/3-1/4-2 mutant system that above-mentioned SD-CMV is infected carries out 5 ' RACE experiment, behind nest-PCR, check order, sequencing result and SD-CMV RNA3 3 ' UTR are carried out sequence alignment, the point of contact that obtains is marked among Fig. 3 with the black hollow arrow, in whole 21 clones, obtain 16 different point of contacts altogether, wherein there are 15 point of contacts to be positioned at 5 ' end of upstream, TLS zone, have only a point of contact to be positioned at the TLS district, and the shearing focus that obtains in 9 point of contacts being arranged and the SD-CMV natural infection being tested overlaps, in addition, comprehensive natural infects experiment and infects the point of contact that the dcl2-1/3-1/4-2 mutant obtains, between Fig. 3 the 76th ~ 80 nt, also can form a little focus, in natural infection 5 ' RACE and mutant 5 ' RACE experiment, detect 3 shearings respectively in this zone.In addition, this result is also shown in and shears the point of contact that the hot spot region obtains may be the shearing product of the RISC of siRNA mediation mostly, explanation owing to there is the higher structure of similar tRNA, make the siRNA-RISC mixture be difficult to enter and these zones of target in the terminal possibility of 3 of CMV 3 ' UTR '.Based on above result, selected some to shear focus and designed amiRNA as the effective target spot of potential.

3) design of amiRNA

The point of contact that obtains in the comprehensive above different experiments, 2051/2052 point of contact, SD-CMV RNA3 3 ' UTR zone most possibly is the shearing focus of siRNA-RISC mediation, because this point of contact has detected respectively in wild-type Arabidopis thaliana and dcl2/3/4 mutant 1 time and 4 times respectively, not only be positioned within " hot spot region ", and the sequence before and after this site is all very conservative between three strain systems of the different subgroups of CMV, one " U " contained at this point of contact in addition, the endonuclease that meets most of miRNA mediation occurs in " U " this rule (Huesken et al., 2005 afterwards to the shearing of target; Reynolds et al., 2004), therefore select this point of contact design amiRNA, called after amiR-SD-3, the sequence of amiR-SD-3 is shown in sequence in the sequence table 1.

In contrast, designed amiR-SD-4, its target spot is between SD-CMV RNA3 2018/2019 Nucleotide, do not detect any point of contact in this site, though in Q-CMV infection wild-type tobacco and SD-CMV infection Arabidopis thaliana, detecting respectively 1 time and 2 shearings between 2017/2018 Nucleotide, and sequence is also more conservative near this point of contact, but does not detect the point of contact in any dcl2/3/4 of coming from mutant.

The amiRNA of embodiment 2, usefulness embodiment 1 cultivates the transgenic plant of cucumber-mosaic-virus resistant

One, the amiRNA of embodiment 1 cultivates the transgenic plant of cucumber-mosaic-virus resistant

A) with the miR159a precursor be the structure of the amiRNA precursor of skeleton

The method of utilizing RT-PCR is template with the arabidopsis thaliana genomic dna, and the precursor 266bp fragment amplification of Arabidopis thaliana miR159a is come out, and this fragment is except containing complete pre-miR159a (184bp), and 5 ' end also comprises the genome sequence of 82bp.RT-PCR the primer miR159-For and miR159-Rev sequence are as follows:

MiR159-For:5 '-

CCACAGTTTGCTTATGTCGGATCC-3 ' and miR159-Rev:5 '-ATG TAGAGCTCCCTTCAATCC-3 '.

Wherein miR159-For primer 5 ' end italicized item is the XbaI site of introducing, and miR159-Rev primer line part is 18 Nucleotide in 21 nucleotide sequences of sophisticated miR159.

The fragment that obtains behind the pcr amplification reclaims the back with gel electrophoresis and is connected with pGEM-T Easy vector (Progema) carrier, selects the plasmid called after pGEM-T Easy-pre-miR159a that the forward clone obtains after sequence verification.Downcut the XbaI-SalI fragment from this plasmid, be connected to through same enzyme and cut on the expression of plants binary vector pCAMBIA-1300221 of digestion, and the precursor sequence of MIR159a is placed the downstream of 35S promoter, called after 35S-pre-miR ¹⁵⁹

In order to make up the amiRNA precursor, designed following primer, used primer is as shown in table 1, wherein, italicized item is the BglII site that carries in the miR159a genome sequence in the Forward primer, underscore is the hairpin structure complementary strand of the amiRNA of structure partly, has also introduced XbaI (italic) site in addition in amiR-SD-3 and amiR-SD-4 Forward primer.And underscore is partly represented in the Reverse primer is the reverse fully-complementary sequence of ripe amiRNA sequence.

Table 1, amiRNA primer

With pGEM-T Easy-pre-miR159a plasmid is template, this carries out pcr amplification to primer with the Forward of amiR-SD-3 and Reverse, the fragment that obtains is connected on pGEM-T Easy vector (Progema) carrier, called after pGEM-T Easy-pre-amiR-SD-3, check order, sequencing result shows, the nucleotides sequence of the DNA of coding amiRNA precursor is classified the sequence 2 in the sequence table as, the amiRNAs precursor that the DNA of coding amiRNA precursor transcribes out, the same with the precursor of natural MIR159a, it is the single stranded RNA that in plant, can produce Microrna, this single stranded RNA forms loop-stem structure, the nucleotide sequence that contains sequence 1 in the ordered list in the fragment of the stem of its loop-stem structure, the stem of its loop-stem structure has two base pairs not complementary, this not two base pairs of complementary corresponding to sequence 1 from 5 ' terminal the 9th and the 10th.

With pGEM-T Easy-pre-miR159a plasmid is template, this carries out pcr amplification to primer with the Forward of amiR-SD-4 and Reverse, the fragment that obtains is connected on pGEM-T Easy vector (Progema) carrier, called after pGEM-T Easy-pre-amiR-SD-4.

The amiRNA precursor fragment that pGEM-T Easy-pre-amiR-SD-3 and pGEM-T Easy-pre-amiR-SD-4 obtain after Bgl II and SpeI digestion is cloned into the 35S-pre-miR that cuts digestion through same enzyme ¹⁵⁹On the plasmid, with the precursor of amiRNA precursor replacement miR159a, the recombinant plasmid called after 35S-pre-amiR-SD-3 and the 35S-pre-amiR-SD-4 that obtain, wherein the amiRNA precursor sequence is positioned at the downstream of 35S promoter, just obtained the expression vector of amiRNA thus, as shown in Figure 5.

B) acquisition of the transgenic plant of cucumber-mosaic-virus resistant

Respectively 35S-pre-amiR-SD-3 and 35S-pre-amiR-SD-4 are changed in Arabidopis thaliana (Columbia) and the tobacco (Xanthi.nc).Utilize " flower is contaminated " method to carry out the conversion (Clough and Bent, 1998) of Arabidopis thaliana.Tobacco transform then adopt with Agrobacterium altogether cultured method carry out, with the MS resistance substratum screening transfer-gen plant that contains the 20mg/L Totomycin.The Arabidopis thaliana that 4 weeks are big or small and the tobaccos of 3～4 all sizes are used for the virus infection experiment.

Two, transgenic plant are to the resistance analysis of CMV

A) transgenic arabidopsis is to the resistance analysis of CMV

SD-CMV virus (Du, Q.S., C.G.Duan, Z.H.Zhang, Y.Y.Fang, R.X.Fang, Q.Xie, and H.S.Guo.2007.DCL4 targets Cucumber mosaic virus satelliteRNA at novel secondary structures.J.Virol.81:9142-51) (Institute of Microorganism, Academia Sinica) to infect the method for experiment as follows:

1) infection there is the blade of CMV place aseptic precooling mortar, adds 5mMNaHPO4 (pH7.2) damping fluid (1g adds 5 mlNaHPO4 damping fluids as the fresh blade in malicious source) of certain volume in proportion, grind homogenate with mortar;

2) before the inoculation, will wait to inoculate evenly spray cloth silicon carbide of plant leaf surface;

When 3) inoculating, dip in the contrary leaf cant of taking juice to the blade that rubs gently, 2～3 blades of every strain plant inoculation with finger;

4) inoculation back 10min sprays clear water to blade surface, washes residual silicon carbide;

5) postvaccinal plant moisturizing is one day, cultivates under 16hr illumination/8hr dark condition then, and culture temperature is 23 ℃, symptom occurs after about 2 weeks.

SD-CMV infects wild-type Arabidopis thaliana (Col-0) can cause serious developmental malformation, as downgrade, internode apart from shorten, inflorescence and the bending of fruit pod etc.The T2 that select to change amiRNA gene Arabidopis thaliana carries out SD-CMV and attacks the poison experiment for (comprising and isozygoty and heterozygote) 3 the strains systems (Fig. 7 A) with high amiRNA expression level in the plant, is contrast with the wild-type." amiR-SD-3 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-3 among Fig. 7 A, and " amiR-SD-4 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-4.

Infected back 12 days, and had 26 strains typical susceptible symptom to occur in the 27 strain wild-type Arabidopis thalianas, similarly, most of Arabidopis thaliana that changes 35S-pre-amiR-SD-4 also shows susceptible symptom (Fig. 7 B).With the Arabidopis thaliana that changes 35S-pre-amiR-SD-4 forms sharp contrast is that all plants that change three strains systems of 35S-pre-amiR-SD-3 (1,2,7) all present normal growth phenotype." WT " represents the wild-type Arabidopis thaliana that SD-CMV infects among Fig. 7 B; " mock " represents the wild-type Arabidopis thaliana of uninfecting virus, and " amiR-SD-3 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-3, and " amiR-SD-4 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-4.

Do enzyme linked immunological experiment (ELISA) subsequently again and detect to have infected the proteic accumulating level of SD-CMV CP in back 16 days system's leaves.Utilize the proteic polyclonal antibody serum of SD-CMV CP (Qu, J., J.Ye, and R.Fang.2007.Artificial microRNA-mediated virus resistance in plants.J.Virol.81:6690-6699) (Microbe Inst., Chinese Academy of Sciences) carries out the ELISA experiment and detects the proteic accumulation of SD-CMV CP.The goat anti-rabbit immunoglobulin of alkali phosphatase enzyme mark is as two anti-(available from Amersham Biosciences).CP protein accumulation level by protein extract and pNPP substrate colour developing 1hr after absorbance value at the 405nm place react, detecting instrument is Microplate Reader (BIO-RAD, Model 550).

The result shown in Fig. 7 C, SD-CMV CP in changeing 35S-pre-amiR-SD-3 strain system, detect less than, consistent with observed phenotype.Change the SD-CMV CP albumen that has then accumulated higher level in the 35S-pre-amiR-SD-4 strain system leaf, similar to the wild-type plant that SD-CMV infects, " SD-CMV " represents the Arabidopis thaliana that SD-CMV infects among Fig. 7; " mock " represents the Arabidopis thaliana of uninfecting virus, and WT is the wild-type Arabidopis thaliana, and " amiR-SD-3 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-3, and " amiR-SD-4 " represents the Arabidopis thaliana of 35S-pre-amiR-SD-4.

It is the proteic change dynamics of SD-CMV CP in the transgenic plant that T3 generation of having done simultaneously that second independent experiment monitor that SD-CMV infects isozygotys, as shown in table 2, change the resistance of 35S-pre-amiR-SD-4 strain system and also have only 18.8-28.6%, similar to T2 for the resistance result of plant.But the transgenic plant that change 35S-pre-amiR-SD-3 keep 100% resistance to SD-CMV.What Fig. 7 D showed is the change dynamics of SD-CMV CP albumen in system's leaf, the transgenic plant SD-CMV CP in detection period that changes 35S-pre-amiR-SD-3 maintains background level all the time, and the transgenic plant of commentaries on classics 35S-pre-amiR-SD-4 are after infecting, SD-CMV CP level sharply increased in preceding 8 days, maintained higher level in 8 ~ 16 days, similar to the wild-type contrast.

Among Fig. 7 D, the wild-type Arabidopis thaliana that " WT/Mock " representative is infected with clear water, the wild-type Arabidopis thaliana that on behalf of SD-CMV, " WT/SD-CMV " infect, amiR-SD-3/SD-CMV " the amiR-SD-3 transgenic arabidopsis of representing SD-CMV to infect; the amiR-SD-4 transgenic arabidopsis that on behalf of SD-CMV, " amiR-SD-4/SD-CMV " infect, X-coordinate are the fate (dpi) after SD-CMV infects plant.

Comprehensive two groups of result of experiment, but target spot is positioned at the amiR-SD-3 of the possible RISC entry site that detects maximum number of times resistance the best to SD-CMV, and the resistance of amiRNA mediation can heredity.

Table 2.AmiRNA transgenic arabidopsis is for the resistance of SD-CMV

Two groups of independent experiments of expression detect the resistance of amiRNA transgenic arabidopsis to SD-CMV in the table, first group of experiment for T2 for transfer-gen plant, second group of experiment for T3 for the pure and mild system of transgenosis.* molecule is represented the ELISA positive plant among the I/T, and denominator is represented infected plant sum; * R represents resistance per-cent.

B) transgene tobacco is to the resistance analysis of CMV

SD-CMV, Fny-CMV (Rizzo, T.M.and Palukaitis, P.Nucleotide sequence andevolutionary relationships of cucumber mosaic virus (CMV) strains:CMV RNA1.J.Gen.Virol.1989 70 (1): 1-11) (Institute of Microorganism, Academia Sinica) and Q-CMV virus (Davies, C.and Symons, R.H.Further implications for the evolutionaryrelationships between tripartite plant viruses based on cucumber mosaicvirus RNA 3.Virology.1988,165 (1): 216-24.) (Institute of Microorganism, Academia Sinica) infects the same step a) of method of experiment.

Metainfective tobacco is cultivated under 16hr illumination/8hr dark condition, and culture temperature is 25 ℃.

Tobacco is the natural host of CMV, and Q-CMV infects tobacco can classical symptoms such as piebald occur at system's leaf usually.And SD-CMV (subgroup IB) and Fny-CMV (subgroup IA) symptom are stronger, comprise the serious chlorosis of system's blade, distortion etc.

At first detecting changes the resistance of amiRNA genetic tobacco to Q-CMV.According to the resistance result of transgenic arabidopsis, the strain system (Fig. 8 A) that 3 of tobacco T1 generations of select changeing 35S-pre-amiR-SD-3 have an amiRNA high level expression carries out Q-CMV and attacks the poison experiment.Infected back 6 days, and had 14 strains tangible susceptible symptom (Fig. 8 B) to occur in the 15 strain wild-type tobaccos, the tobacco T1 that changes 35S-pre-amiR-SD-3 tangible infection symptoms do not occur in generation.

ELISA detects the accumulating level of Q-CMV CP albumen in system's leaf, utilizes the proteic polyclonal antibody serum of Q-CMV CP.The proteic polyclonal antibody serum of Q-CMV CP is prepared as follows:

With pQCD3 (Ding, S.W., J.P.Rathjen, W.X.Li, R.Swanson, H.Healy, and R.H.Symons.1995.Efficient infection from cDNA clones of cucumbermosaic cucumovirus RNAs in a new plasmid vector.J.Gen.Virol.76 (Pt2): 459-464) (Institute of Microorganism, Academia Sinica) is template, with QCP-anbibody-5 and QCP-antibody-3 is that primer carries out pcr amplification and goes out Q CMV CP gene, and the primer sequence of QCP-anbibody-5 and QCP-antibody-3 is as follows: GGATCCatggacaaatctggatctcc and GCGGCCGCCtaagtcgggagcatccgtgag cuts rear clone to prokaryotic expression carrier pGEX-4T-2 with BamHI and NotI enzyme after order-checking is identified, obtain pGEX-4T-2-QCP prokaryotic expression recombinant plasmid.The pGEX-4T-2-QCP recombinant plasmid is changed among the bacillus coli DH 5 a, selecting positive colony contains in the LB liquid nutrient medium of penbritin to 2ml, 18～25 ℃ of jolting to OD600 values are about 0.6, this 2ml bacterium liquid is added among the 100ml LB, 18～25 ℃ of jolting to OD600 values are about 0.6, adding IPTG then is that 0.1～1mM continues to shake 5～8h to final concentration, 4 ℃, the centrifugal 5min of 5000rpm collects thalline, (adds 1%Triton-100 with the PBS of 10 * column volume, proteinase inhibitor) suspension thalline, ultrasonic disruption cell in the frozen water (1 minute/time * 5 times, each 1 minute at interval) or twice of high pressure fragmentation, 4 ℃, 12, the centrifugal 15min of 000rpm, collect supernatant liquor, add DTT to its final concentration be 1mM, use the 0.45um filter paper filtering, filtrate was used for the sweet peptide agarose affinity chromatography of Guang post.

Affinity column prepares with the following method:

1) with GST-glutathione agarose gel (Beijing ancient cooking vessel state Bioisystech Co., Ltd) mixing, get 1.5ml and add in the chromatography column, add 10ml 20% ethanol, make agarose natural subsidence in post;

2) after 10ml 20% ethanol flows to end, add 10ml PBS and clean pillar (5 * column volume), when treating that the PBS liquid level did not just have gel in the pipe, put the cover of drip, stand-by;

3) supernatant liquor after the filter adds affinity column, makes mixed solution naturally by chromatography column under the room temperature, keeps the 0.5ml filtered liquid and does PAGE electrophoresis detection usefulness;

4) 10ml PBS washes pillar 3 times, closes at every turn and collects washings 0.5ml survey OD value when finishing;

5) 3 * column volume elutriant (the reduced glutathion solution of 10mM) eluted protein, every pipe is collected the 0.5ml elutriant;

6) the elutriant protein concentration is respectively managed in survey;

7) PAGE electrophoresis detection purity;

Obtain the proteic polyclonal antibody serum of Q-CMV CP of rabbit behind the Q-CMV CP protein immunization rabbit with purifying.

Carry out the ELISA experiment and detect the proteic accumulation of Q-CMV CP.The goat anti-rabbit immunoglobulin of alkali phosphatase enzyme mark is as two anti-(available from Amersham Biosciences).CP protein accumulation level by protein extract and pNPP substrate colour developing 1hr after absorbance value at the 405nm place embody, detecting instrument is MicroplateReader (BIO-RAD, Model 550).

Experimental result shows, in the wild-type plant system leaf that Q-CMV infects, can detect the proteic a large amount of accumulation of CP (Fig. 8 C), (lines 6 and change 35S-pre-amiR-SD-3,15 and 18) all plants all show normal phenotype, and detect less than the proteic accumulation of Q-CMV CP (Fig. 8 B, C, table 3).

Table 3:amiRNA transgene tobacco T1 is for the resistance analysis that Q-CMV is infected

In the table 3 among the I/T molecule represent the ELISA positive plant, denominator is represented infected plant sum; R represents resistance per-cent.

Having carried out two groups subsequently independently tests to have detected and changes the resistance of the tobacco of 35S-pre-amiR-SD-3 to SD-CMV.Infect the tobacco that changeed 35S-pre-amiR-SD-3 in back 6 days and shown the height resistance that SD-CMV is infected, 35S-pre-amiR-SD-3 strain system ( lines 6,15 and 18) all plants all show normal growth phenotype (Fig. 8 D); The proteic accumulation detection method of SD-CMV CP is with embodiment 4, and the proteic accumulation of SD-CMV CP also detects less than (Fig. 8 E) in system's leaf.In second group of experiment, also obtained similar result, as shown in table 4.The tobacco of two groups of experiment transfer 35S-pre-amiR-SD-3 is 91.7-100% to the resistance per-cent of SD-CMV, and is similar to the resistance result who obtains in Arabidopis thaliana.

Two groups of resistances of independently testing the detection transgene tobacco have also been done simultaneously to Fny-CMV, because Fny-CMVCP albumen and SD-CMV CP aminoacid sequence height similar (98.2%), therefore SD-CMV CP polyclonal antibody serum is also adopted in its accumulation, and method as described in example 4 above.

Experimental result is shown in Fig. 8 F and 8G, because Fny-CMV and SD-CMV 3 ' UTR nucleotide sequence height similar (88.9%), and belong to subgroup I together, in two groups of experiments transgenic plant with quite similar to the resistance of SD-CMV, the tobacco that promptly changes 35S-pre-amiR-SD-3 presents the height resistance to infecting of Fny-CMV to the resistance of Fny-CMV.More than the symptom phenotype of the transgenic plant of Jian Ceing is observed altogether and is continued 2 months.

Table 4.amiRNA transgene tobacco T1 is for the resistance analysis to SD-CMV and Fny-CMV

In the table 4 among the I/T molecule represent the ELISA positive plant, denominator is represented infected plant sum; R represents resistance per-cent.

Among Fig. 8 B, D and the F, the wild-type Arabidopis thaliana that on behalf of SD-CMV, " WT " infect; " mock " represents the wild-type Arabidopis thaliana of uninfecting virus; Among Fig. 8 A, C, E and the G, the tobacco of 35S-pre-amiR-SD-3 is changeed in " amiR-SD-3 " representative, " mock " represents the tobacco of uninfecting virus, " Q-CMV " represents the tobacco of Q-CMV virus infection, " SD-CMV " represents the tobacco of SD-CMV virus infection, " Fny-CMV " represents the tobacco of Fny-CMV virus infection, and WT is a wild-type tobacco.

The specificity analyses of the amiRNA of embodiment 3, embodiment 1 in transgenic plant

For the transgenosis stably express of getting rid of amiRNA non-specific silence to plant endogenous gene, promptly so-called " missing the target " phenomenon, the sequence input NCBI BLAST website of all amiRNA is compared, found that, in the 21nt of amiRNA sequence, can surpass 15 nt with the continuous complementary pairing of the endogenous transcript of Arabidopis thaliana without any an amiRNA.In order further to get rid of the possibility of amiRNA targeted plants native gene, simultaneously with amiRNA sequence input " Plant miRNA Target Finder "

( Http:// bioinfo3.noble.org/miRNA/miRU.htm) " TIGR Arabidopsis GeneIndex 1 " and " TIGR Tobacco Gene Index 1 " database carries out the search of potential target gene.The result shows, in Arabidopis thaliana, have only an endogenous transcript-AT3G29250-to form and be less than 3 incomplete pairing with amiR-SD-3, comprise mispairing and disappearance, as shown in Figure 6A, and in tobacco, do not detect any one endogenous transcript and the amiRNA sequence is less than 3 mispairing.Whether can reduce the level of Arabidopis thaliana AT3G29250 mRNA for the transgene expression that detects amiR-SD-3, total RNA with Arabidopis thaliana wild-type and amiR-SD-3 transgenic plant is a template, design a pair of primer and carry out the experiment of sxemiquantitative reverse transcription PCR, level with the actin transcript is contrast, and primer sequence is as follows:

AT3G29250-forward ATGTCGGGGCTAAGGGAAGTTT

AT3G29250-reverse TGATACGTCCAGAGGTGCTCTT

The result is shown in Fig. 6 B, show in the amiR-SD-3 transgenic plant, the expression level of AT3G29250 transcript is compared not obviously downward modulation with the wild-type contrast, illustrates that the transgene expression of amiR-SD-3 can not influence the expression of the endogenous AT3G29250 of Arabidopis thaliana.

Sequence table

＜110〉Institute of Microorganism, Academia Sinica

＜120〉silencing complex mediated shearing site and the application thereof of RNA inductive

<130>CGGNARW81317

<160>2

<210>1

<211>21

<212>RNA

＜213〉cucumber mosaic virus (Cucumber mosaic virus)

<400>1

ccacgacuga?ccauuuuagc?c 21

<210>2

<211>184

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

gggctaaaat?ggaaagtcgt?ggcatgagtt?gagcagggta?aagaaaagct?gctaagctat 60

ggatcccata?agccctaatc?cttgtaaagt?aaaaaaggat?ttggttatat?ggattgcata 120

tctcaggagc?tttaacttgc?cctttaatgg?cttttactct?tcccacgact?gaccatttta 180

gccc 184

Claims (9)

1. strain is the application of artificial Microrna in cucumber-mosaic-virus resistant of genomic 3 ' UTR zone the 2051/2052nd site design at cucumber mosaic virus SD;

The nucleotides sequence of described artificial Microrna is classified sequence 1 in the sequence table as.

2. artificial Microrna, it is characterized in that: the nucleotides sequence of described artificial Microrna is classified sequence 1 in the sequence table as.

3. the encoding gene of the described artificial Microrna of claim 2, be that to comprise cucumber mosaic virus SD strain be the 2051/2052nd site, genomic 3 ' UTR zone to coding, and with the cucumber mosaic virus genome complementation of these both sides, site, length is the dna molecular of the RNA of 21nt.

4. the encode dna molecular of artificial Microrna precursor, it is characterized in that: the nucleotide sequence of described dna molecular is shown in sequence in the sequence table 2.

5. the recombinant expression vector that contains the described dna molecular of claim 4.

6. the transgenic cell line that contains the described dna molecular of claim 4.

7. the reorganization bacterium that contains the described dna molecular of claim 4.

8. a method of cultivating the plant of cucumber-mosaic-virus resistant is that the described dna molecular of claim 4 is changed in the plant, obtains the transgenic plant of cucumber-mosaic-virus resistant.

9. method according to claim 8 is characterized in that: described plant is tobacco or Arabidopis thaliana.

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