CN1706939A - Antinuclear polyhedron virus transgenic silkworm and generating method thereof - Google Patents
Antinuclear polyhedron virus transgenic silkworm and generating method thereof Download PDFInfo
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Abstract
The aim of the invention is to provide a transgenic silkworm exhibiting a resistance against nucleopolyhedrovirus (NPV) and a method for producing the same. This transgenic silkworm holding a DNA encoding an RNA molecule having an RNAi effect against a gene indispensable for the proliferation of the NPV, as capable of expressing is formed. As a result of the examination of the proliferation of the NPV in the transgenic silkworm, it is elucidated that the proliferation of the virus can be inhibited.
Description
Technical field
The present invention relates to the transgenic silkworm of antinuclear polyhedron virus.In addition, the present invention relates to the method for the transgenic silkworm of producing antinuclear polyhedron virus.
Background technology
At silkworm industry workshop, silkworm is by the serious infringement of nuclear polyhedrosis virus (NPV).Up to now, many investigators are carrying out producing the research work of anti-NPV silkworm, and still, none is achieved success so far.This be since in the world existing silkworm all lack anti-NPV gene.In order to stop the infringement of this virus, egg-incubation district and relevant device are carried out thorough disinfection encouraged to use.Yet, not only bother in a such way of breeding silkworms, and caused huge manpower and materials consumption, the cost costliness of cocooing, and become one of complicated factor in farm.
NPV has at least 50 or the big DNA C-type virus C of more a plurality of genes.As everyone knows, these genes are bred expression in host silkworm cell.Known wherein some genes are necessary (the Kool M of viral growth, AhrensCH, Goldbach RW, Rohrmann GF, Vlak JM. (1994) Identification of genes involved inDNA replication of the Autographa californica baculovirus.Proc.Natl.Acad.Sci.USA91 (23): 11212-11216; Lu A, Miller LK. (1995) The roles of eighteen baculovirus lateexpression factor genes in transcription and DNA replication.J.Virol.69 (2): 975-982).
Recently, having developed some utilizes transposon or marker gene to produce the novel method (Tamura of transgenic silkworm, T. (1999) Methods for producing transformed silkworms using transposons.Abstracts of the7th Conference on Insect Functions, p10-22; Horn, C., B, Jaunich and E.A.Wimmer, (2000) Highly sensitive, fluorescent transformation marker for Drosophila transgenesis.Dev Genes Evol 210:623-629; Horn, C., and E.A.Wimmer, (2000) A versatile vetor setfor animal transgenisis Dev Genes Evol 210:630-637; Thomas, J.L., M.Da Rocha, A.Besse, B.Mauchamp and G.Chavancy, 2002 3xP3-EGFP marker facilitates screening fortransgenic silkworm Bombyx mori L.from the embryonic stage onwards.Insect BiochemMol Biol 32:247-253; Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G, Shirk, P., Fraser, M., Prudhomme, J.-C.and Couble, P. (2000) A piggyBac element-derived vector efficientlypromotes germ-line transformation in the silkworm Bombyx mori L.NatureBiotechnology 18,81-84; Berghammer, A.J., Klingler and E.A.Wimmer, (1999) Auniversal marker for transgenic insects.Nature 402:370-371; Tomita, M., Sato, T., Adachi, T., Munetsuna, H., Tamuara, T., Kanda, T., Yoshizato, K. (2001) Production of humancollagen gene-incorporated transgenic silkworm.Abstracts of the 24th Annual Meeting ofMolecular Biology Society of Japan).These methods use DNA type piggyBac transposon as carrier, use green fluorescent protein (GFP) the gene gene that serves as a mark.Because these methods can effectively be produced recombinant chou, therefore applied to multiple practical applications, for example be used to produce useful albumen (Tomita, M., M..H., SatoT, Adachi T, Hino R, Haysshi M, Shimizu K, Nakamura N, Tamura T, Yoshizato, K., (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons.Nat.Biotechnol.21,52-56.) and new fiber, and cultivate the mutation that is considered to have ntiviral characteristic.Therefore, this class research of a part has been set about carrying out.
Summary of the invention
In view of said circumstances has been made the present invention.The purpose of this invention is to provide a kind of transgenic silkworm of anti-NPV resistance and method of production thereof of showing.More particularly, a kind of NPV-resistant transgenic silkworm that carries the expression type DNA of coding RNA molecule is provided, this RNA molecule demonstrates the RNAi effect (RNAi effect) for the essential gene (agene essential for proliferation of NPV in silkworms) of NPV propagation, and the production method of this silkworm.
The inventor has carried out macromethod makes it to have reached above-mentioned purpose.At first, the contriver has checked the silkworm that whether can produce anti-NPV by transgenic silkworm.More specifically, can form the gene of the mRNA with hair fastener type structure as the template synthetic, and detect the propagation situation of NPV in the transgenic silkworm that carries this gene with the gene that derives from virus.The result is that viral proliferation is significantly suppressed.
More specifically, the present invention relates to show the transgenic silkworm of anti-NPV resistance and the method for production thereof.[1]-[17] is specific purposes of the present invention, is described below.
[1] show the transgenic silkworm of antinuclear polyhedron virus resistance, wherein this silkworm carries the expression type DNA of a kind of RNA molecule of coding, and this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene.
[2] as the transgenic silkworm of [1], wherein the DNA of coding RNA molecule is the DNA that a kind of coding has the RNA molecule of hair fastener type structure.
[3] as [2] described transgenic silkworm, wherein to have the DNA of RNA molecule of hair fastener type structure be a kind of like this DNA to coding, wherein the encode adopted coding DNA of having of adopted RNA being arranged and has adopted coding DNA complementary DNA to be connected of the arbitrary zone of mRNA by joint with this, make described have adopted coding DNA and complementary DNA thereof mutually towards, wherein said mRNA coding nuclear polyhedrosis virus is bred necessary gene.
[4] as each described transgenic silkworm of [1]-[3], it is the lef1 gene that wherein said nuclear polyhedrosis virus is bred necessary gene.
[5] as the transgenic silkworm of [4], coding have the DNA of adopted RNA to be (a) or DNA (b) wherein:
(a) contain the DNA of nucleotide sequence shown in the SEQ ID NO:1;
(b) contain the DNA of nucleotide sequence shown in the SEQ ID NO:1, the Nucleotide of wherein one or more is substituted, and deletion is inserted, and/or adds.
[6] as the transgenic silkworm of [4], wherein to demonstrate the DNA of the RNA molecule of RNAi effect be the DNA that comprises the nucleotide sequence shown in the SEQ ID NO:2 to coding.
[7] a kind of production shows the method for the transgenic silkworm of antinuclear polyhedron virus resistance, and this method may further comprise the steps:
(a) will encode a kind of DNA of RNA molecule changes silkworm seed over to, and this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene;
(b) from the silkworm that the ovum that has changed described DNA over to is hatched, pick out transgenic silkworm.
[8] method of the silkworm of preparation antinuclear polyhedron virus, wherein this method has comprised the step of the DNA that expresses a kind of RNA molecule of coding in the silkworm cell, this RNA demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene.
[9] as the method for [7] or [8], wherein the DNA of coding RNA molecule is the DNA that a kind of coding has the RNA molecule of hair fastener type structure.
[10] as [9] described method, wherein to have the DNA of RNA molecule of hair fastener type structure be a kind of like this DNA to coding, wherein the encode adopted coding DNA of having of adopted RNA being arranged and has adopted coding DNA complementary DNA to be connected of the arbitrary zone of mRNA by joint with this, make described have adopted coding DNA and complementary DNA thereof mutually towards, wherein said mRNA coding nuclear polyhedrosis virus is bred necessary gene.
[11] as each method of [7]-[10], it is the lef1 gene that wherein said nuclear polyhedrosis virus is bred necessary gene.
[12] include the carrier of the DNA of a kind of RNA molecule of encoding, this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene, and wherein said DNA is inserted between the inverted terminal repeat sequence of transposon and with promoter function and links to each other; Described DNA comprises that coding has adopted coding DNA of having of adopted RNA and a complementary DNA thereof, and this has the have adopted RNA of adopted RNA for the arbitrary zone of mRNA of the described gene of coding; Wherein said have adopted coding DNA and its complementary DNA by joint be connected make they mutually towards.
[13] as the carrier of [12], it is the lef1 gene that described nuclear polyhedrosis virus is bred necessary gene.
[14] as the carrier of [13], coding have the DNA of adopted RNA to be (a) or DNA (b) wherein:
(a) contain the DNA of nucleotide sequence shown in the SEQ ID NO:1;
(b) contain the DNA of nucleotide sequence shown in the SEQ ID NO:1, the Nucleotide of wherein one or more is substituted, and deletion is inserted, and/or adds.
[15] as the carrier of [13], wherein to demonstrate the DNA of the RNA molecule of RNAi effect be the DNA that comprises the nucleotide sequence of SEQID NO:2 to coding.
[16] test kit, the carrier that it comprises each described carrier of [12]-[15] and has the DNA of coding transposase.
[17] have the reagent of silkworm of preparation antinuclear polyhedron virus, this reagent contains each described carrier of [12]-[15], the perhaps test kit of [16].
Description of drawings
Fig. 1 represents to be used to produce the carrier structure of transgenic silkworm.P represents to derive from the SK18 promotor of virus, lef represents the part lef1 gene relevant with virus replication (direction of representing gene with arrow), Fbp (A) is the poly a-signal of silk fibroin gene source, and A3pGFP represents to be used to distinguish the marker gene of transgenic silkworm.Having length between adopted DNA of having of lef1 and antisense DNA is the joint of 96 Nucleotide.
Fig. 2 is illustrated in the viral proliferation in the transgenic silkworm with antiviral gene.The virus of some amount (each individual 105 viral polyhedron) is applied give transgenic silkworm (tg) and control group after after 72 hours and 96 hours, the viral load in the mensuration blood.Viral load is to measure by the method for the real-time quantitative viral DNA of (real-time) PCR.
Embodiment
The present invention is the expression of the DNA by the inducing a kind of coding RNA molecule silkworm that produced anti-NPV successfully first, and wherein the RNA molecule has demonstrated the RNAi effect of NPV being bred necessary gene in the silkworm cell.
The present invention is based on this research, and a kind of transgenic silkworm that shows anti-NPV resistance is provided, and this transgenic silkworm carries the expression type DNA of coding RNA molecule, and this RNA molecule demonstrates the RNAi effect of anti-NPV propagation indispensable gene.
In the present invention, the implication that " carries the transgenic silkworm of the expression type DNA of coding RNA molecule; this RNA molecule demonstrates the RNAi effect of NPV being bred necessary gene " not only comprises the transgenic silkworm of this DNA of constitutive expression, but also is included in the transgenic silkworm that can express this DNA under the actual conditions (as inducible expression).
The DNA of the RNA molecule of coding performance RNAi effect of the present invention has comprised that those coding metabolites show the DNA (for example product that obtains by the cracking of RNA chain) of the RNA molecule of RNAi effect.
Coding of the present invention shows as the DNA of the RNA molecule of RNAi effect, and preferably coding has the DNA of the RNA molecule of hair fastener type structure.The example of DNA that coding has a RNA molecule of hair fastener type structure includes, but not limited to contain the DNA that justice (or antisense) codon DNA is arranged that justice (or antisense) RNA is arranged in the arbitrary zone of coding mRNA, the propagation indispensable gene of this mRNA coding NPV; And with this DNA complementary DNA by joint be connected make mutually towards.
Here, " mutually towards " means that two sequences is positioned at opposed facing direction each other.In other words the structure that DNA of the present invention has is that its inverted terminal repeat sequence that has is to have the DNA of adopted RNA to form by connect coding with joint.In particular, for example be when coding has the dna sequence dna (two strands) of adopted RNA:
AGTC (sense strand)
::::
TCAG (antisense strand)
Above-mentioned by joint form inverted terminal repeat sequence, coding has the structure of the dna sequence dna of adopted RNA to be expressed as:
AGTC-LLL-GACT
:::: ::::
TCAG-LLL-CTGA
(" L " represents any Nucleotide of joint here, ": " expression hydrogen bond)
The structure that DNA of the present invention preferably has is as above-mentioned structure, the arbitrary zone of target gene mRNA of promptly encoding the adopted coding DNA of having of adopted RNA arranged, make them be connected towards ground mutually with the complementary DNA of this DNA by joint." complementary DNA " can be described as the DNA of encoding antisense in the RNA in above-mentioned " the arbitrary zone of target gene mRNA " more specifically.Therefore, in other words, DNA of the present invention can be described as having such structure, wherein has encoding antisense and connects with relative (opposite) direction by joint with this DNA complementary DNA in the antisense code dna of the RNA in the arbitrary zone of target gene mRNA.
Because DNA of the present invention has the joint between inverted repeats, DNA transcription product so of the present invention also has the structure that is included in the joint in the inverted terminal repeat sequence.And the RNA molecule with this spline structure forms hydrogen bond usually and forms hairpin structure between tumor-necrosis factor glycoproteins.
The DNA length of integral connector of the present invention is not limited especially, only needs it can make contiguous tumor-necrosis factor glycoproteins form hydrogen bond.Yet, when intron not being counted, its length range usually at several Nucleotide (as 1-10 Nucleotide) between about 100 Nucleotide, tens Nucleotide (as 60,70,80, or 90 Nucleotide) more preferably.
The dna nucleotide sequence of integral connector is not limited especially, can be nucleotide sequence arbitrarily.And as long as can form hydrogen bond between the above-mentioned inverted repeats, joint is not absolute demand yet.Therefore, DNA of the present invention has comprised the situation that lacks joint.
Adopted coding DNA is arranged, or the length of its complementary DNA in DNA of the present invention, 1000 Nucleotide or still less normally, more preferably about 500 Nucleotide.
And in the present invention, the RNA base pair in the double-stranded RNA part does not need to match fully, in fact can exist unpaired site.The unpaired site that exists in does not to a certain degree influence RNA and forms.
DNA of the present invention can use common genetic engineering technique synthetic by those skilled in the art.For example, can use following operation preparation: from 5 ' terminal the 430th Nucleotide of the ORF sequence of lef1, and from 5 ' terminal the 526th Nucleotide, carry out pcr amplification respectively, amplified production is linked together with relative direction (opposite orientations).
For example, NPV propagation indispensable gene of the present invention has comprised the lef1 gene, ie-1 gene, and p143 (dna helicase) gene.The nucleotide sequence of these genes has been disclosed in Accession No:NC_001962, the lef1 gene, the ie-1 gene, with the p143 gene be respectively ORF6 (GENE ID:1488636), ORF123 (GENE ID:1488755), and ORF78 (GENE ID:1724488) (Sumiko Gomi, Kei Majima and SusumuMaeda, Journal of General Virology (1999), 80,1323-1337).
Those skilled in the art know viral DNA and are easy to variation.Thereby gene of the present invention is not limited in the gene that contains above-mentioned open sequence, as long as described mutator gene is essential for NPV propagation.
When the lef1 gene is selected as NPV propagation indispensable gene, for example the DNA of coding RNA molecule contains has adopted coding DNA to comprise to contain the DNA of SEQ ID NO:1 sequence, but is not limited thereto.One or more Nucleotide are substituted, and deletion is inserted, and/or the DNA that contains SEQ ID NO:1 sequence that adds can be utilized too.
Comprise the replacement of one or more Nucleotide, deletion is inserted, and/or the sudden change in the SEQ ID NO:1 nucleotide sequence that adds, and has comprised that not only spontaneous mutation has also comprised artificial mutation.The sudden change quantity and the mutational site of nucleotide sequence are unrestricted, as long as this nucleotide sequence can be expressed and be shown the RNA molecule of nuclear polyhedrosis virus being bred the RNAi effect of necessary gene.Usually, sudden change quantity is no more than 10% of total nucleic acid, preferably is no more than 5%, more preferably no more than 1%.
When the lef1 gene is selected as target gene, for example the DNA example of RNA molecule of coding with hair fastener type structure comprised that (there is adopted sequence in 1-430:lef1 site, position to the DNA that contains SEQ ID NO:2 sequence, position 431-526: joint sequence, position 527-956: antisense sequences), but not as limit.
The present invention produces the method for transgenic silkworm and will describe in an embodiment; Yet transgenic silkworm of the present invention is not restricted to the silkworm that produces with the described method of embodiment.
For example, transgenic silkworm of the present invention can be by such method production: change the DNA of coding RNA molecule over to silkworm seed, wherein the RNA molecule demonstrates the RNAi effect of antinuclear polyhedron virus NPV propagation indispensable gene, picks out transgenic silkworm from the silkworm that the ovum that carries described DNA is hatched.
For example, DNA is changed over to the following enforcement of silkworm seed: the piggyBAC transposon is seted out as vector injection educate early stage silkworm seed (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C.and Couble, P., 2000, Nature Biotechnology 18,81-84).
For example, carrier (the Handler that contains the DNA between the transposon inverted terminal repeat sequence, A.M., McCombs, S.D., Fraser, M.J., Saul, S.H. (1998) Proc.Natl.Acad.Sci.U.S.A.95 (13): 7520-5), wherein this DNA has such DNA, and its coding demonstrates the RNA of the RNAi effect of target NPV propagation institute indispensable gene, and functional downstream that is connected to any promotor, this carrier is transferred to silkworm seed together with the carrier (assistant carrier) of the DNA that carries the coding transposon.
In the present invention, " functional connection " expression promotor is connected with DNA, thereby makes transcription factor and promotor in conjunction with inducing the DNA that is positioned at the promotor downstream to express.Thereby, as long as DNA transcribes and can take place, between promotor and this DNA, any DNA sequence can be arranged so.
For example, the promotor that is fit to the present invention's use comprises, but be not restricted to, the promotor of the gene that can in organizing arbitrarily, express, as the SK18 promotor, the promotor of the promotor of heat shock protein gene and kytoplasm Actin muscle and tRNA gene, the heterologous gene promoter systems as the GAL4/UAS system that utilizes the transcriptional control system and in fatty body (fat body) and midgut the promotor of specific expressed gene.
Further, the example of assistant carrier comprises, but is not restricted to pHA3PIG (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E, Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, Shirk, P., Fraser, M., Prudhomme, J.-C.and Couble, P., 2000, NatureBiotechnology 18,81-84), but not as limit.
The example of transposon of the present invention comprises piggyBac, but not as limit, mariner and minos are used (Shimizu, K., Kanba, M., Sonobe, H., Kanda, T., Klinakis, A.G., Savakis, C.andTamura, T. (2000) Insect Mol.Biol., 9,217-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).
In the present invention, baculovirus vector also can be used to production transgenic silkworm of the present invention (Yamao, M., N.Katayama, H.Nakazawa, M.Yamakawa, Y.Hayashi et al., 1999, Genes Dev.13:511-516).
Below, describe the method that DNA is changed over to silkworm seed in detail; But, of the present inventionly change DNA the method for silkworm seed over to not as limit.For example, can DNA be injected into silkworm seed with one and change dna direct over to silkworm seed with needle tubing.In preferred embodiments, beat a hole with physics or chemical process in advance on eggshell, DNA changes silkworm seed over to by this hole exactly.In this embodiment, be used for by the insertion angle that this hole is injected into DNA the tubular stinger of ovum should keep with silkworm seed abdomen surface near vertical.
In the present invention, carrying out the method for physics punching on eggshell, for example is with pin or microlaser.Preferably, use pin on eggshell, to punch.The manufactured materials of pin, the intensity of pin etc. do not need special qualification, as long as this pin can punch on silkworm seed and just can.The pin that the present invention uses is normally with the shaft-like pin that a tip is arranged: still, have more than and be defined as this shape.As long as can punch on silkworm seed, the object of Any shape can adopt.For example, " pin " of the present invention also comprised having most advanced and sophisticated pyramid object, cuspidated circular cone object.In the present invention, preferably use the tungsten pin.The hole that the thickness (diameter) of the pin that the present invention adopts is got wants to allow following kapillary pass, thereby the thickness of pin is usually greatly about 2-20 μ m, or 5-10 μ m preferably.In addition, the method for chemically punching on eggshell can adopt chemical substance, includes but not limited to hypochlorous acid.
In the present invention, special restriction is not done in the position of punching, as long as can make the insertion angle of the tubular stinger by this hole injection DNA keep approaching vertical with silkworm seed abdomen surface.The optimum seeking site ground of punching at the ventral surface (ventral surface) of ovum or its to the side, preferred in ventral surface, even more preferably slightly towards the back side end (back end) of silkworm seed ventral surface central authorities.
Among the present invention, " near vertical " means 70 °-120 ° more preferably 80 °-90 °.Among the present invention, " will grow and be paotoblastic position " usually presses close to the position (normally distance ovum surface 0.01mm-0.05mm position) on ovum surface at the ovum veutro, preferably in the outside of belly central authorities of ovum, near the ovum surface location slightly by the position of rear end.
The pipe that the present invention injects the DNA employing is not particularly limited material, intensity, interior diameter etc.; Yet, before inserting, the DNA syringe on eggshell, punches with physics or chemical process, and the thickness (outside diameter) of preferred pipe should be wanted to pass the hole that this is opened.For example, the example of such pipe comprises, but is not defined as glass capillary.
The preferred embodiment of DNA transfer method in the present invention, utilization has been equipped the mechanical manipulator (manipulator) of pin and DNA syringe and has been carried out following steps, on silkworm seed, punch with physics or chemical process, the DNA syringe is passed this hole insert silkworm seed, it is approaching vertical with the silkworm seed outside of belly that it inserts angle, injection DNA.Usually, the present invention preferably uses to comprise with above-mentioned mechanical manipulator and operates as the device of building block.
The building block of such device comprises, dissecting microscope, light source, moveable platform, is fixed to microscopical thick mechanical manipulator, is fixed on mechanical arm and the syringe that can regulate DNA injection air pressure on the thick mechanical manipulator with metal parts.The pressure that syringe uses is provided by the nitrogen jar, and uses push-switch to open pressure valve.The silkworm seed of injecting is fixed on the matrix, glass slide for example, and the position of silkworm seed is moved and is then used moveable platform control.The controller that connects four pipes is used for the glass capillary of operation micromachined hand.Actual mechanical process is, uses thick mechanical manipulator that the tungsten pin is aimed at silkworm seed, moves horizontally silkworm seed by the platform control stick and punches.The controller of following operation micromachined hand makes the tip of glass capillary be directed to the position of punching, re-uses the platform control stick kapillary is inserted silkworm seed.In this scheme, glass capillary must insert and make it vertical with the silkworm seed outside of belly.Open push-switch injection DNA, the manipulation bar is extracted kapillary again.Use quick-acting tackiness agents or analogue to seal this hole, in the hatcher of fixed temperature and humidity, look after silkworm seed.The device that the present invention uses is the described device of Japanese Patent NO.1654050 preferably, or its improvement product.
Further, in concrete scheme of the present invention, the silkworm seed that is used as the DNA transfer preferably is fixed in the substrate (substrate).Be fit to substrate of the present invention and for example comprise slide and plastic sheet, but not as limit.In concrete scheme of the present invention, ideal situation is that fixing after the direction of silkworm seed is appropriately placed, making DNA can be injected into growth accurately is paotoblastic silkworm seed position.Further, in concrete scheme, be fixed to suprabasil silkworm seed quantity and be not particularly limited.In the time will operating a plurality of silkworm seed, by its orientation preferably being fixed on silkworm seed in the substrate so that the abdomen of silkworm seed back of the body placement direction should be consistent.The present invention can adopt fixedly, and the method for silkworm seed has, for example, induce silkworm to lay eggs on a kind of paper of buying that is wiped with water soluble glue in advance (dredging ovum card (loose egg cards)), on paper, separate ovum by adding water, then wet ovum is lined up row in substrate, air-dry.Preferably be fixed on the slide with suitable steering handle ovum.And, can use double sticky tape, analogues such as tackiness agent are fixed on ovum in the substrate.
The method example whether inspection DNA is transferred to silkworm seed comprises, for example, brings up again the DNA that gets injection and measured (Nagaraju, J., Kanda, T., Yukuhiro.K., Chavancy, G, Tamura, T. ， ﹠amp from ovum; Couble, P. (1996) .Attempt of transgenesis of the silkworm (Bombyx mori L) by egg-injection offoreign DNA.Appl.Entomol.Zool., 31,589-598), perhaps observe genetic expression (Tamura, T., the Kanda of the DNA that is injected into silkworm seed, T., Takiya, S., Okano, K. ， ﹠amp; Maekawa, H. (1990) .Transientexpression of chimeric CAT genes injected into early embryos of the domesticatedsilkworm, Bombyx mori.Jpn.J.Gemet., 65,401-410).
Produce in the method for transgenic silkworm in the present invention, following step relates to: pick out transgenic silkworm from the silkworm of the egg-incubation that changed DNA of the present invention over to.For example, in the present invention, can pick out transgenic silkworm by using the selection markers thing.Being used for selective marker of the present invention, is the normally used marks of those skilled in the art, comprises, but be not defined as that fluorescin is CFP for example, GFP (as A3GFP), YFP, and DsRed.Adopt these marks, can use the fluorescent and stereo microscope to detect transgenic silkworm easily.And, because the difference of fluorescence color can be used multiple mark simultaneously.
The present invention also further provides the carrier that is used for aforesaid method, and test kit.Particularly, the invention provides the carrier of the DNA that contains a kind of RNA molecule of encoding, this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene.In preferred concrete scheme, described DNA is inserted into the inverted terminal repeat sequence of transposon, be connected with promoter function ground, and comprising that coding has the complementary DNA of the adopted coding DNA of having of adopted RNA and this DNA, the described adopted RNA that has that adopted RNA is arranged for the arbitrary zone of mRNA of this gene of coding; Described have adopted coding DNA and complementary DNA thereof to connect towards ground mutually by joint.For example such carrier comprises, but be not defined as, contain the carrier that is connected to as the DNA in promotor downstreams such as SK18, this DNA also is inserted into the transposon inverted terminal repeat sequence (Tamura that derives from the piggyBac carrier, T., et al., 2000, Nature Biotechnology 18,81-84), wherein the DNA of Cha Ruing has coding the DNA of justice (or antisense) coding DNA and the complementary DNA of this DNA is arranged, the former encode NPV propagation indispensable gene coding mRNA arbitrary zone justice (or antisense) RNA arranged, described have adopted coding DNA and complementary DNA thereof to connect by joint so that its mutually towards.Selected as the propagation of the NPV in carrier indispensable gene when the lef1 gene, the DNA that contains aforementioned sequence is used as has adopted coding DNA and coding to have the DNA of the RNA molecule of hair fastener type structure.The present invention also provides the test kit that contains above-mentioned carrier and the carrier (assistant carrier) of the DNA with coding transposon.For example assistant carrier comprises, but is not defined as, and pHA3PIG (Tamura, T., et al., 2000, NatureBiotechnology 18,81-84).And, the invention provides the purposes of these carriers and test kit.Especially, the invention provides the purposes as the reagent that produces anti-NPV silkworm, this reagent contains carrier of the present invention or test kit as activeconstituents.
The purposes of the present invention on industry is to provide the transgenic silkworm that demonstrates anti-NPV by the transgenic silkworm that recombinant viral genome is carried in generation.
Producing the very big benefit that shows anti-NPV silkworm is to avoid the most serious problem, the i.e. infringement of NPV in the silkworm industry workshop.Thereby this can improve the efficient of silkworm variety production, saves the farming labor force, and produces high quality silk.
The patent of quoting among the application, patent application and public publication are hereby incorporated by.
Embodiment
The present invention can explain in detail with reference to following examples; Yet, not as limit.
In order to suppress viral growth, a kind of gene that derives from virus is used for recombinant production can prepares gene with hairpin structure mRNA.The gene of producing and the structural table of carrier are shown among Fig. 1.
The lef1 gene is the gene relevant with the dna replication dna of NPV in the silkworm, also is the necessary gene of virus multiplication.Connect this gene, wherein this genetic transcription direction is reversed, and is used in the culturing cell the strong active SK18 of performance as its promotor.And, can carry the transgenic silkworm of this gene with differentiation with the A3GFP gene gene that serves as a mark, A3GFP can reach fluorescin at whole silkworm body surface, and can be easy to tell the individuality that carries this gene at the fluorescent and stereo microscopically.
With this carrier DNA and can express and have the DNA (auxiliary DNA) of the factor that target gene is imported to the effect of goal gene group and be injected into and grow in the early stage silkworm seed.The larva that raising hatches from ovum grows for adult up to it, obtains the s-generation then.Identify that by under fluorescent microscope, observing these s-generation larvas whole silkworm body sends the individuality of fluorescence.Those look like the individuality of transgenic silkworm, will be continued to raise and architecture.Give be in behind the last worm transgenic silkworm in the length of time 72 hours, 96 hours of these architectures with the NPV polyhedron of some amount, measure virus multiplication in the blood by PCR in real time.The result shows, in carrying the silkworm of gene as shown in Figure 1, the propagation of virus is subjected to obvious inhibition as shown in Figure 2.
The above results shows, the generation of carrying the transgenic silkworm by making the gene that the virogene reorganization produced can cause the generation of anti-virus type silkworm.
Sequence table
＜110〉Japan as Represented by Director General of Ministry of Agriculture, Forestry an (NATIONAL INSTITUTE OF AGROBOLOGICALSCIENCES)
＜120〉transgenic silkworm of antinuclear polyhedron virus and production method thereof
<130>MOA-A0408
<160>2
<170>PatentIn?version?3.1
<210>1
<211>430
<212>DNA
<213>Bombyx?mori
<400>1
atgttattgt?gcaattatac?acagaagcgc?gtcgacatga?tgtgggacgc?gattgcgtac 60
aacgacagcc?gcaagtacgc?gttcatgacg?gtcaacgcgc?gctggattca?cgccgacaaa 120
tattttgata?ccgccgcaca?attgtatagt?tacattgtgc?aaaacaaagt?gtccgacgtg 180
cacgtcaaac?cgttggacga?cggcggcggc?agggaatggg?tcgtagacgc?cgattacaag 240
aattatgttg?acgaacacga?tttaatgctg?aaaatttaca?ttggcgccac?ggcgtttttg 300
ttgttttaca?cggaagagaa?cgtgtcaaga?gtcatgtat?accggcaaccg?tggatttcac 360
ttgtggttaa?aattcaccga?caagtttaaa?atcacgtccg?ctcaaaatgt?tcgcgtgcat 420
cggtaca?agg 430
<210>2
<211>956
<212>DNA
＜213〉artificial
<220>
＜223〉dna sequence dna of synthetic
<400>2
atgttattgt?gcaattatac?acagaagcgc?gtcgacatga?tgtgggacgc?gattgcgtac 60
aacgacagcc?gcaagtacgc?gttcatgacg?gtcaacgcgc?gctggattca?cgccgacaaa 120
tattttgata?ccgccgcaca?attgtatagt?tacattgtgc?aaaacaaagt?gtccgacgtg 180
cacgtcaaac?cgttggacga?cggcggcggc?agggaatggg?tcgtagacgc?cgattacaag 240
aattatgttg?acgaacacga?tttaatgctg?aaaatttaca?ttggcgccac?ggcgtttttg 300
ttgttttaca?cggaagagaa?cgtgtcaaga?gtcatgtata?ccggcaaccg?tggatttcac 360
ttgtggttaa?aattcaccga?caagtttaaa?atcacgtccg?ctcaaaatgt?tcgcgtgcat 420
cggtacaagg?tatgcggaat?gtacaaacgt?acggcctctc?tcacacaatg?cgcaaaactg 480
cccggctgaa?tgcaatcact?atccaacttt?gcaggttttt?cgaaggcctt?gtaccgatgc 540
acgcgaacat?tttgagcgga?cgtgatttta?aacttgtcgg?tgaattttaa?ccacaagtga 600
aatccacggt?tgccggtata?catgactctt?gacacgttct?cttccgtgta?aaacaacaaa 660
aacgccgtgg?cgccaatgta?aattttcagc?attaaatcgt?gttcgtcaac?ataattcttg 720
taatcggcgt?ctacgaccca?ttccctgccg?ccgccgtcgt?ccaacggttt?gacgtgcacg 780
tcggacactt?tgttttgcac?aatgtaacta?tacaattgtg?cggcggtatc?aaaatatttg 840
tcggcgtgaa?tccagcgcgc?gttgaccgtc?atgaacgcgt?acttgcggct?gtcgttgtac 900
gcaatcgcgt?cccacatcat?gtcgacgcgc?ttctgtgtat?aattgcacaa?taacat 956
Claims (17)
1, show the transgenic silkworm of antinuclear polyhedron virus resistance, wherein this silkworm carries the expression type DNA of a kind of RNA molecule of coding, and this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene.
2, transgenic silkworm as claimed in claim 1, wherein the DNA of coding RNA molecule is the DNA that a kind of coding has the RNA molecule of hair fastener type structure.
3, transgenic silkworm as claimed in claim 2, wherein to have the DNA of RNA molecule of hair fastener type structure be a kind of like this DNA to coding, wherein the encode adopted coding DNA of having of adopted RNA being arranged and has adopted coding DNA complementary DNA to be connected of the arbitrary zone of mRNA by joint with this, make described have adopted coding DNA and complementary DNA thereof mutually towards, wherein said mRNA coding nuclear polyhedrosis virus is bred necessary gene.
4, as arbitrary described transgenic silkworm among the claim 1-3, wherein to breed necessary gene be the lef1 gene to nuclear polyhedrosis virus.
5, transgenic silkworm as claimed in claim 4, wherein coding have the DNA of adopted RNA to be (a) or DNA (b):
(a) contain the DNA of the nucleotide sequence of SEQ ID NO:1;
(b) contain the DNA of the nucleotide sequence of SEQ ID NO:1, the Nucleotide of wherein one or more is substituted, and deletion is inserted, and/or adds.
6, transgenic silkworm as claimed in claim 4, wherein to demonstrate the DNA of RNA molecule of RNAi effect be the DNA that contains the nucleotide sequence of SEQ ID NO:2 to coding.
7, a kind of generation shows the method for the transgenic silkworm of antinuclear polyhedron virus resistance, and this method may further comprise the steps:
(a) will encode a kind of DNA of RNA molecule changes silkworm seed over to, and wherein the RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene; With
(b) from the silkworm that the ovum that has changed described DNA over to obtains, filter out transgenic silkworm.
8, a kind of method that is used to prepare the silkworm of antinuclear polyhedron virus, wherein this method has comprised the step of the DNA that expresses a kind of RNA molecule of coding in the silkworm cell, this RNA demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene.
9, as claim 7 or 8 described methods, wherein the DNA of coding RNA molecule is the DNA that a kind of coding has the RNA molecule of hair fastener type structure.
10, method as claimed in claim 9, wherein to have the DNA of RNA molecule of hair fastener type structure be a kind of like this DNA to coding, wherein the encode adopted coding DNA of having of adopted RNA being arranged and has adopted coding DNA complementary DNA to be connected of the arbitrary zone of mRNA by joint with this, make described have adopted coding DNA and complementary DNA thereof mutually towards, wherein said mRNA coding nuclear polyhedrosis virus is bred necessary gene.
11, as each described method of claim 7-10, it is the lef1 gene that wherein said nuclear polyhedrosis virus is bred necessary gene.
12, the carrier that includes the DNA of a kind of RNA molecule of encoding, this RNA molecule demonstrates the RNAi effect of nuclear polyhedrosis virus being bred necessary gene, and wherein said DNA is inserted between the inverted terminal repeat sequence of transposon and with promoter function and links to each other; Described DNA comprises that coding has adopted coding DNA of having of adopted RNA and a complementary DNA thereof, and this has the have adopted RNA of adopted RNA for the arbitrary zone of mRNA of the described gene of coding; Wherein said have adopted coding DNA and its complementary DNA by joint be connected make they mutually towards.
13, carrier as claimed in claim 12, it is the lef1 gene that wherein said nuclear polyhedrosis virus is bred necessary gene.
14, carrier as claimed in claim 13, wherein coding have the DNA of adopted RNA to be (a) or DNA (b):
(a) contain the DNA of the nucleotide sequence of SEQ ID NO:1;
(b) contain the DNA of the nucleotide sequence of SEQ ID NO:1, the Nucleotide of wherein one or more is substituted, and deletion is inserted, and/or adds.
15, carrier as claimed in claim 13, the DNA that demonstrates RNAi effect RNA molecule that wherein encodes is the DNA that contains nucleotide sequence shown in the SEQ ID NO:2.
16, test kit, the carrier that it includes each described carrier of claim 12-15 and has the DNA of coding transposase.
17, a kind of reagent that is used to prepare the antinuclear polyhedron virus silkworm, this reagent contain each described carrier of claim 12-15, the perhaps described test kit of claim 16.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP159121/04 | 2004-05-28 | ||
| JP2004159121A JP2005333913A (en) | 2004-05-28 | 2004-05-28 | Transgenic silkworm exhibiting resistance against nucleopolyhedrovirus and method for producing the same |
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| Publication Number | Publication Date |
|---|---|
| CN1706939A true CN1706939A (en) | 2005-12-14 |
| CN100567481C CN100567481C (en) | 2009-12-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB2005100792590A Expired - Fee Related CN100567481C (en) | 2004-05-28 | 2005-05-30 | The transgenic silkworm of antinuclear polyhedron virus and production method thereof |
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| Country | Link |
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| JP (1) | JP2005333913A (en) |
| CN (1) | CN100567481C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660157A (en) * | 2018-05-30 | 2018-10-16 | 苏州大学 | A kind of vitro construction method of the silkworm cytoplasmic polyhedrosis virus based on DNA vector |
| CN111004852A (en) * | 2020-01-03 | 2020-04-14 | 江苏科技大学 | Method for screening resistance of bombyx mori nuclear polyhedrosis virus by using BmIML-2 gene |
| CN111471714A (en) * | 2020-05-07 | 2020-07-31 | 西南大学 | Eukaryotic transgenic cell line mediated by Minos transposon system and construction method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103687949A (en) * | 2011-02-04 | 2014-03-26 | N·加瓦哥瓦达 | Virus resistant transgenic silkworm |
| CN114836419A (en) * | 2022-04-11 | 2022-08-02 | 浙江洪晟生物科技股份有限公司 | RNA molecule for preventing and treating bombyx mori nuclear polyhedrosis virus and bombyx mori antiviral preparation thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4132760B2 (en) * | 2001-09-19 | 2008-08-13 | 独立行政法人農業生物資源研究所 | Efficient introduction method of polynucleotides into silkworm eggs |
-
2004
- 2004-05-28 JP JP2004159121A patent/JP2005333913A/en active Pending
-
2005
- 2005-05-30 CN CNB2005100792590A patent/CN100567481C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660157A (en) * | 2018-05-30 | 2018-10-16 | 苏州大学 | A kind of vitro construction method of the silkworm cytoplasmic polyhedrosis virus based on DNA vector |
| CN108660157B (en) * | 2018-05-30 | 2020-12-04 | 苏州大学 | DNA vector-based in vitro construction method of bombyx mori cytoplasmic polyhedrosis virus |
| CN111004852A (en) * | 2020-01-03 | 2020-04-14 | 江苏科技大学 | Method for screening resistance of bombyx mori nuclear polyhedrosis virus by using BmIML-2 gene |
| CN111004852B (en) * | 2020-01-03 | 2023-07-25 | 江苏科技大学 | Method for screening silkworm nuclear polyhedrosis virus disease resistance by using BmIML-2 gene |
| CN111471714A (en) * | 2020-05-07 | 2020-07-31 | 西南大学 | Eukaryotic transgenic cell line mediated by Minos transposon system and construction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100567481C (en) | 2009-12-09 |
| JP2005333913A (en) | 2005-12-08 |
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