阅读说明：本技术 早熟禾半潜隐病毒作为vigs载体的应用 (Application of half cryptovirus of annual bluegrass as VIGS carrier ) 是由 张永亮 李召雷 李大伟 胡佳成 姜志豪 于嘉林 王献兵 韩成贵 王颖 于 2019-05-21 设计创作，主要内容包括：本发明提供早熟禾半潜隐病毒作为VIGS载体的应用。所述基因沉默载体系统包括含有早熟禾半潜病毒RNAα、RNAβ和RNAγ表达盒的质粒；其中,RNAα、RNAβ和RNAγ分别位于不同的表达盒中,这些表达盒位于同一质粒或位于不同质粒上；所述RNAγ表达盒中还含有靶标序列,所述靶标序列位于RNAγ中γb蛋白编码基因的3`端。本发明基于早熟禾半潜病毒的基因沉默载体系统可以对谷子等单子叶植物的基因组进行高效的基因沉默。(The present invention provides application of half cryptovirus of annual bluegrass as VIGS carrier.The gene silencing vector system includes the plasmid containing annual bluegrass viral RNA α partly latent, RNA β and RNA γ expression cassette；Wherein, RNA α, RNA β and RNA γ are located in different expression cassettes, these expression cassettes are located at same plasmid or are located on different plasmids；Also contain target sequence in the RNA γ expression cassette, the target sequence is located at the end 3` of γ b protein coding gene in RNA γ.The present invention is based on the gene silencing vector systems of half occult virus of annual bluegrass can carry out efficient gene silencing to the monocotyledonous genome such as millet.)

1. following any application of half cryptovirus of annual bluegrass (Poa semilatent virus):

1) it is used for VIGS carrier；

2) it is used for gene silencing.

2. a kind of gene silencing vector system based on half occult virus of annual bluegrass, which is characterized in that the gene silencing vector system System includes the plasmid containing annual bluegrass viral RNA α partly latent, RNA β and RNA γ expression cassette；

Wherein, RNA α, RNA β and RNA γ are located in different expression cassettes, these expression cassettes are located at same plasmid or are located at On different plasmids；

Also contain target sequence in the RNA γ expression cassette, the target sequence is located at 3 of γ b protein coding gene in RNA γ The end `.

3. gene silencing vector system according to claim 2, which is characterized in that the expression cassette is driven by T7 promoter Expression.

4. gene silencing vector system according to claim 2, which is characterized in that the carrier that sets out of the plasmid is selected from pBluescript II SK(+)、pMD19-T。

5. any one of the claim 2-4 gene silencing vector system is carrying out the application in gene silencing to plant.

6. application according to claim 5, which is characterized in that the plant is monocotyledon.

7. application according to claim 6, which is characterized in that the monocotyledon includes millet, barley, wheat, jade Rice.

8. application according to claim 7, which is characterized in that using the gene silencing vector system to millet PDS base Because carrying out gene silencing.

9. application according to claim 8, which is characterized in that the gene silencing vector system includes:

(1) pBluescriptII SK (+) plasmid containing RNA α；

(2) the pMD19-T plasmid containing RNA β；And

(3) pBluescript of barley PDS gene target sequence is integrated with containing RNA γ and at the end γ b protein coding gene 3` II SK (+) plasmid；

Wherein, for expanding the PCR primer of barley PDS gene target sequence are as follows:

F10:5 '-ctctgactgcagaggTCAATGTTCATATATGGTTTG-3 '；

R10:5 '-agtactcatatgaggGCAGCGATTTCATCTGGAAAC-3 '.

10. the biomaterial containing any one of the claim 2-4 gene silencing vector system, which is characterized in that the life Object material includes transposons, plasmid vector, phage vector, viral vectors or engineering bacteria.

Technical field

The invention belongs to field of biotechnology, specifically, being related to half cryptovirus of annual bluegrass answering as VIGS carrier With.

Background technique

Virus induced gene silencing (Virus-induced gene silencing, VIGS) carrier is functional genome Research provides strong tool.Before the appearance of VIGS technology, the function of some gene is depended on more traditional Genetic transforming method, usually using being largely mutated in the genome of the radom insertions such as T-DNA or transposons to plant Body, by screening the mutant for obtaining target gene and being knocked (knock-out), and then the phenotype by observing the mutant becomes Change the function to determine target gene.This method can make target gene complete deactivation, there is a good effect, but this method Also have the shortcomings that obvious, not only need to carry out genetic transformation, while needing to screen purpose mutant, complicated for operation, time-consuming, and And if target gene belongs to some gene family, the redundancy on other gene functions in family may be covered in phenotype The missing of this gene.In addition, for certain lethal type genes of development, this method can not obtain its corresponding deletion mutant. These all limit application of this method in functional genome research.On the contrary, VIGS operation is fairly simple, routine is overcome Time-consuming and is difficult to the disadvantages of operating for genetic transformation, and the function of certain target genes can be rapidly and efficiently analyzed by this technology Can, or even can be used for high-throughput functional genome research, by the way that a large amount of cDNA segments in library are inserted into VIGS carrier, It is inoculated with plant using these obtained recombinant viruses, the function of these cDNA segments is judged by observation phenotype.VIGS carrier In the research for having been widely used for plant function gene.

Although being developed to VIGS carrier there are many virus, in the research for plant function gene, for single The gene functional research of cotyledon (monocots) especially gramineae plant, selectable VIGS carrier is still very limited , the monocotyledonous VIGS carrier that can be used for registered at present is mainly derived from following seven virus: barley stripe mosaic Viral (Barley stripe mosaic virus, BSMV), bromovirus (Brome mosaic virus, BMV) are built Clover mosaic virus (Cymbidium mosaic virus, CymMV), rice tungro bacilliform virus (Rice tungro Bacilliform virus, RTBV), bamboo mosaic virus and its satellite RNA (Bamboo mosaic virus together With its associated satellite RNA, BaMV), cucumber mosaic virus (Cucumber mosaic virus, CMV) and Chinese pennisetum mosaic virus (Foxtail mosaic virus, FoMV), each all has some advantages, but also same When come with some shortcomings place.Common problem is symptom after virus infection often disturb target gene silencing phenotype Observation.Some viruses such as BMV after being integrated with target gene, infects and target gene segment often occurs in host processes It loses, so that target gene can not be in whole plant by lasting silencing.In addition, after some viruses infect host such as BSMV itself Certain disease resistance response can be excited, which has limited it to study the application in certain specific passageways genes such as disease-resistant gene function.Though Right above-mentioned seven kinds of virus has been developed to VIGS carrier, but for certain specific industrial crops such as millets, it is selectable VIGS tool only has FoMV at present, and symptom of the FoMV on millet is relatively heavy.Based on this, continually developing new can be used for grass The VIGS carrier of section's plant gene function research, to avoid or improve the defect of existing gramineae plant VIGS carrier, have Important practice significance.

Summary of the invention

Application the object of the present invention is to provide half cryptovirus of annual bluegrass as VIGS carrier.

Present inventive concept is as follows: it is rod-shaped that half occult virus of annual bluegrass (Poa semilatent virus, PSLV) belongs to plant A member of Viraceae (Virgaviridae) Hordeivirus (Hordeivirus), it is more that existing report shows that it can be infected Kind of monocotyledon, the virus have exploitation into the innate advantage of VIGS carrier, and infecting after plant is symptom of hiding, and symptom is very Gently, this observation for being very beneficial for silencing phenotype.The present invention develops PSLV for VIGS carrier, is important monocotyledon such as paddy The functional gene research of son provide it is new can selection tool.

In order to achieve the object of the present invention, in a first aspect, the present invention provides half cryptovirus of annual bluegrass (Poa semilatent Virus following any application):

1) it is used for VIGS carrier；

2) it is used for gene silencing.

Second aspect, the present invention provide a kind of gene silencing vector system based on half occult virus of annual bluegrass, the gene Silent carrier system includes the plasmid containing annual bluegrass viral RNA α partly latent, RNA β and RNA γ expression cassette；

Wherein, RNA α, RNA β and RNA γ are located in different expression cassettes, these expression cassettes be located at same plasmid or On different plasmids；

Also contain target sequence in the RNA γ expression cassette, the target sequence is located at γ b encoding histone base in RNA γ The end 3` of cause.

Preferably, the expression cassette is driven by T7 promoter and is expressed.

It is highly preferred that the plasmid is the plasmid containing T7 promoter, T7 promoter can be such that the geneome RNA of PSLV obtains It is come out with correct transcription, and there is infectivity, corresponding host plant can be infected.

The carrier that sets out of the plasmid is selected from pBluescript II SK (+), pMD19-T etc..These plasmids can pass through quotient Industry approach is bought.

Half cryptovirus of annual bluegrass is more seperated RNA virus, and genome includes three justice, single-stranded geneome RNA, difference Referred to as RNA α, RNA β and RNA γ.

Wherein, the γ a albumen of the α a albumen of RNA α coding and RNA γ coding constitutes the replicase of virus；RNA β coding disease The coat protein CP and triplet motor protein TGBs of poison；RNA γ also encodes a little albumen γ b, it is the base of encoding viral Because of silencing suppressor.When RNA α, RNA β, RNA γ are existed simultaneously, virus can be multiple in host plant under appropriate environment System and movement；When only RNA α and RNA γ are existed simultaneously, virus can replicate in host plant under appropriate environment. PSLV can be infected including barley, wheat, corn, a variety of monocotyledons including millet etc..

The study found that exogenous sequences can be inserted without influencing virus originally in the γ b gene downstream of half cryptovirus of annual bluegrass The duplication and movement of body.Therefore, when required target sequence to be incorporated into RNA γ, in order to make the target gene sequence of integration Column do not influence the system motion of virus, and the present invention selects target sequence needed for being inserted into the downstream of γ b ORF.

It will be appreciated by those skilled in the art that the length of target sequence can be adjusted according to experiment effect.In this hair In bright technical solution, the downstream of target sequence upstream and/or target sequence in addition on half cryptovirus genome of annual bluegrass itself Except sequence, additional sequence can also be had, can be another or multiple target sequences and/or other sequences.

The third aspect, the present invention provide the gene silencing vector system and are carrying out the application in gene silencing to plant.

Application above-mentioned, the plant are monocotyledon, such as millet, barley, wheat, corn.

In the specific embodiment of the present invention, using the gene silencing vector system to millet PDS gene into Row gene silencing.The gene silencing vector system includes:

(1) pBluescript II SK (+) plasmid containing RNA α；

(2) the pMD19-T plasmid containing RNA β；And

(3) millet PDS gene target sequence is integrated with containing RNA γ and at the end γ b protein coding gene 3` PBluescript II SK (+) plasmid.

Plasmid pBluescript II SK (+) and pMD19-T includes a multiple cloning sites, and by inverse PCR and together The geneome RNA of RNA α and the RNA γ of half cryptovirus of annual bluegrass are cloned into pBluescript by the mode of source recombination respectively On II SK (+), RNA β is cloned on pMD19-T, product is referred to as pT7-PSLV α, pT7-PSLV γ and pT7-PSLV β.

Wherein, the PCR primer for expanding millet PDS gene target sequence is (SEQ ID NO:1-2):

F10:5 '-ctctgactgcagaggTCAATGTTCATATATGGTTTG-3 '；

R10:5 '-agtactcatatgaggGCAGCGATTTCATCTGGAAAC-3 '.

Fourth aspect, the present invention provide the biomaterial for containing the gene silencing vector system, the biomaterial packet Include but be not limited to transposons, plasmid vector, phage vector, viral vectors or engineering bacteria.

Further, the present invention is described as follows for target sequence integration involved in technical solution:

Target gene sequence needed for the present invention is perhaps synthesized by PCR amplification simultaneously passes through digestion connection or homologous recombination The methods of, target sequence is cloned into above-mentioned specific position.

Specifically, viral vectors is linearized by inverse PCR first, by expanding or synthesizing target gene sequence And in the sequence homologous with viral vectors of its both ends addition 20bp or so, and then target sequence is cloned by recombining reaction On viral vectors, insertion point is after the ORF of γ b.The both ends of target sequence can also add additional sequence, so-called volume Outer sequence can be the sequence on viral vectors, be also possible to other sequences.It is of course also possible to be connected by such as digestion The methods of target sequence is cloned on viral vectors.

Gene silencing vector system of the present invention can be realized to monocotyledonous gene silencing, and the prior art is overcome Middle hordeivirus (Barley stripe mosaic virus, BSMV), bromovirus (Brome mosaic Virus, BMV), cymbidium mosaic virus (Cymbidium mosaic virus, CymMV), rice tungro bacilliform virus (Rice Tungro bacilliform virus, RTBV), bamboo mosaic virus and its satellite RNA (Bamboo mosaic virus Together with its associated satellite RNA, BaMV), cucumber mosaic virus (Cucumber mosaic Virus, CMV) gene silencing vector may not apply to the defect and deficiency of millet；Simultaneously compared to the gene silencing based on FoMV Carrier, virus itself only causes unconspicuous symptom, and does not influence the growth and development and normal phenotype of host plant itself, thus Be conducive to the Phenotypic Observation after target gene silencing.

5th aspect, the present invention provide the method that a kind of pair of plant carries out gene silencing, to utilize base of the present invention Because silent carrier system carries out.

Gene silencing system of the present invention can import plant tissue by the method that product frictional inoculation is transcribed in vitro In cell, and can be in barley, wheat, corn be replicated in the plants such as millet and movement；It is described to be based on half cryptovirus of annual bluegrass Gene silencing vector, the sequence in sequence comprising target gene may be implemented to plant millet etc. after being inoculated with host plant The silencing of target gene in object.

It is demonstrated experimentally that the method by gene silencing vector system of the present invention by the way that product frictional inoculation is transcribed in vitro After infecting millet, gene silencing can be realized to the endogenous gene of millet, and efficiency can achieve 50% or more.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

The present invention provides a kind of gene silencing vector systems based on half cryptovirus of annual bluegrass, can be by target sequence The downstream of γ b is cloned into without influencing its systemic infection on host plant；It is described based on half cryptovirus of annual bluegrass Gene silencing vector can carry out system silencing to the target gene in host plant.

It compares reported based on hordeivirus (Barley stripe mosaic virus, BSMV), sparrow Wheat mosaic virus (Brome mosaic virus, BMV), cymbidium mosaic virus (Cymbidium mosaic virus, CymMV), rice tungro bacilliform virus (Rice tungro bacilliform virus, RTBV), bamboo mosaic virus and Its satellite RNA (Bamboo mosaic virus together with its associated satellite RNA, BaMV), the gene silencing vector of cucumber mosaic virus (Cucumber mosaic virus, CMV), carrier system of the present invention System can be applied to millet.

The reported gene silencing vector based on FoMV is compared, carrier system of the present invention causes in host plant Symptom milder, so as to do not interfere gene silencing cause Symptom Observation, nor affect on the normal growth of host plant And development.

Detailed description of the invention

Fig. 1 is pT7-PSLV α carrier structure schematic diagram in the embodiment of the present invention 1.

Fig. 2 is pT7-PSLV β carrier structure schematic diagram in the embodiment of the present invention 1.

Fig. 3 is pT7-PSLV γ carrier structure schematic diagram in the embodiment of the present invention 1.

Fig. 4 is the symptom figure and corresponding Western that PSLV infects barley, wheat and millet in experimental example 2 of the present invention Blot detection figure.

Fig. 5 is pT7-PSLV γ MCS carrier structure schematic diagram in the embodiment of the present invention 3.

Fig. 6 is pT7-PSLV γ MCS-GFP in the embodiment of the present invention 3₂₅₅Carrier structure schematic diagram.

Fig. 7 is pT7-PSLV γ MCS-HvPDS in the embodiment of the present invention 3₂₄₇Carrier structure schematic diagram.

Fig. 8 is the gene silencing vector system of 3 mid-early maturity standing grain of experimental example, half cryptovirus of the present invention applied to barley and small The phenotypic map and RT-qPCR of wheat detect figure.

Fig. 9 is that the gene silencing vector system of 3 mid-early maturity standing grain of experimental example, half cryptovirus of the present invention is applied to the table of millet Type figure and RT-qPCR detection figure.

Figure 10 is pT7-PSLV γ MCS-HvIspH in the embodiment of the present invention 4₂₀₅Carrier structure schematic diagram.

Figure 11 is the gene silencing vector system of 4 mid-early maturity standing grain of experimental example, half cryptovirus of the present invention applied to barley and small The phenotypic map and RT-qPCR of wheat detect figure.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Half cryptovirus PSLV strain of annual bluegrass used is by University of California Berkeley in the embodiment of the present invention 1 Andrew professor O.Jackson present.Referring to Slykhuis, J.T. (1972) .Poa semilatent virus from native grasses.Phytopathology 62,508-513.