JP3977189B2 - Gene transfer method to fruit tree using apple latent virus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a vector for introducing a foreign gene into a fruit tree prepared by modifying a latent virus of a fruit tree into a virus vector carrying a foreign gene, and a simple fruit tree using the foreign gene introduction vector. It relates to foreign gene transfer methods.
[0002]
[Prior art]
Conventionally, a transformation method using Agrobacterium (Agrobacterium tumefaciens, Agrobacterium rhizogenes) is generally used as a method for introducing a foreign gene into a plant.
[0003]
[Problems to be solved by the invention]
However, fruit trees such as apples have few successful cases, and no simple method for introducing foreign genes has been established.
The present inventor has collected and purified from a spherical virus having a diameter of about 25 nm (ALSV, apple small spherical latent virus), which is a kind of latent virus that is latently infected without causing disease in apple trees. The amino acid sequence encoded by the single-stranded RNA was determined, and based on the research results of successful cDNA synthesis using the single-stranded RNA as a template, this clone was inoculated into a plant to confirm the infection. The present invention was completed by succeeding in infecting and expressing a foreign gene by inserting it between a protease cleavage sequence of a clone and inoculating a plant.
According to the method of the present invention, it can be actually used as a new means for introducing a foreign gene into a fruit tree which has been difficult in the past and transforming it.
[0004]
[Means for Solving the Problems]
That is, the present invention relates to a vector for introducing a foreign gene into fruit trees, which contains DNA for the genomic RNA of the latent virus of fruit trees such as apples. This vector is an infectious vector that can be infected by inoculating fruit trees.
The present invention further comprises a foreign gene introduction vector composition into fruit trees obtained by introducing a foreign gene into the foreign gene introduction vector, and inoculating the foreign tree with the foreign gene introduction vector composition. The present invention relates to a method for introducing foreign genes into fruit trees. The present invention also relates to a method of inoculating a fruit tree with a foreign gene transfer vector composition, introducing the foreign gene into the fruit tree, and expressing the foreign gene in a plant body (for example, a leaf) of the fruit tree. . In addition, the foreign gene may be expressed at a site different from the site where the plant body is inoculated.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the fruit tree latent virus from which the genomic RNA is derived is not particularly limited, and examples thereof include viruses belonging to the Comoviridae family such as apple small spherical latent virus. In the case of using an apple globules latent virus, a vector containing a DNA for the full length of the genome RNA1 of the apple globules latent virus and a vector containing a DNA for the full length of the genome RNA2 of the apple globules latent virus preferable.
The DNA for the genomic RNA of the latent virus of the fruit tree of the present invention can be easily prepared by those skilled in the art based on the viral genome information using a well-known technique in the technical field. For example, it can be easily prepared as a cDNA obtained using reverse transcriptase. Alternatively, it can be prepared as a synthetic DNA by a chemical synthesis method.
The foreign gene transfer vector thus prepared contains at least a part of the genomic RNA of the fruit tree latent virus, but in order to ensure high infectivity to the fruit tree, the full length of the genomic RNA is required. It is preferable to contain DNA for. Therefore, in such a case, a polyA sequence having an appropriate length (eg, several tens to several hundreds) found in viral genomic RNA is also included.
[0006]
Furthermore, the foreign gene transfer vector of the present invention preferably contains various appropriate transcriptional regulatory factors known to those skilled in the art, such as promoters and enhancers, upstream of the DNA for genomic RNA.
For example, examples of promoters include various promoters derived from appropriate plant viruses such as the 35S promoter derived from cauliflower mosaic virus (CaMV).
[0007]
Furthermore, in the foreign gene transfer vector of the present invention, it is preferable to introduce a new restriction enzyme site as a foreign gene insertion site. Such a restriction enzyme site can be introduced at any location as long as the infectivity to the fruit tree of the foreign gene transfer vector of the present invention is not impaired. That is, in the viral genome RANA, it is preferable that a restriction enzyme site is introduced between the base sequences encoding each protein.
For example, in the case of apple small spherical latent virus, a restriction enzyme site is introduced between the base sequence encoding the intercellular transfer protein (MP) and the base sequence encoding the coat protein (Vp25) in genomic RNA2.
Such restriction enzyme sites can be introduced by an appropriate method well known to those skilled in the art. For example, a base sequence encoding a protease cleavage site in a polyprotein in genomic RNA 2 is repeated, and a restriction enzyme site is introduced therebetween. In order to facilitate the insertion of foreign genes, it is preferable to introduce a plurality of different restriction enzyme sites.
[0008]
The foreign gene introduction vector composition to fruit trees of the present invention comprises a foreign gene introduction vector in which a desired foreign gene is introduced into a restriction enzyme site. This foreign gene transfer vector composition can appropriately contain other components, for example, an appropriate buffer component, depending on the infection method and the infection target.
The content of the foreign gene transfer vector in the composition can be appropriately selected by those skilled in the art depending on the infection method, the infection target, and the like. For example, a foreign gene transfer vector having a DNA concentration in the range of 0.01 to several μg / μl can be contained in a buffer such as Tris-EDTA.
[0009]
The foreign gene transfer vector composition of the present invention can be inoculated to an applicable site of fruit trees by any method known to those skilled in the art as long as the foreign gene is significantly introduced into the fruit trees. For example, fruit trees can be inoculated with a foreign gene transfer vector composition of about several μl to several tens of μl per leaf by a carbon random method which is a general method. Alternatively, it is also possible to inoculate the obtained juice after treating a part of the fruit tree previously infected with the exogenous gene transfer vector composition of the present invention, for example, by suitable means such as grinding. .
[0010]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
[0011]
Example 1
Genomic structure of apple small spherical latent virus, a new virus belonging to the Comoviridae family The small spherical virus used in the present invention (Apple Latent Spherical Virus; “ALSV”) is afflicted with ring-shaped rust A virus isolated from apple trees by Koganase et al. (1985). Subsequent research has revealed that ALSV is not a pathogen of ring-shaped rust, but a virus that can infect apples.
For the purpose of clarifying the taxonomic affiliation of ALSV, the present inventors analyzed the physicochemical properties and genome structure of ALSV, and clarified the classification affiliation of ALSV (Li, Yoshizawa, et al., J of General Virology (2000), 81, 541-547). The results are summarized as follows:
[0012]
Physicochemical properties of ALSV (1) When virus was purified from ALSV-infected Chenopodium quinoa by the method of Koganase et al., Particles were purified as a single peak component after sucrose density gradient centrifugation. The purified sample showed an ultraviolet absorption curve of a typical nucleoprotein having a maximum absorption at 259 nm and a minimum absorption at 240 nm, and A _260/280 was 1.85 to 1.90. In addition, many small spherical viruses with a diameter of 25 nm were observed by electron microscope observation of the purified virus preparation. The yield of purified virus was 0.4-0.6 mg from 100 g of infected leaves.
(2) the purified virus preparation was cesium equilibrium density gradient centrifugation chloride separation, divided into two peaks of buoyant density 1.41 g / cm ³ and 1.43 g / cm ^3, clear that ALSV comprises two particle component Became.
(3) The analysis of the ALSV coat protein (CP) by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and three bands with molecular weights of 25 kilodaltons (KDa), 24 KDa, and 20 KDa were detected. . In Western blot analysis, these proteins reacted with antisera produced by Koganase et al. From the above results, it was revealed that ALSV is composed of three types of CP (Vp25, Vp24, Vp20).
(4) When nucleic acid was extracted from the purified virus using ammonium carbonate buffer and protease K and subjected to 1% agarose gel electrophoresis under formamide denaturation, two bands of about 6800 bases and 3600 bases were detected. These nucleic acids were completely digested with RNase under conditions of 2XSSC and 0.1XSSC. From the above results, it was concluded that the ALSV genome consists of two-segment single-stranded RNA [RNA1 (6800 bases) and RNA2 (3600 bases)].
[0013]
ALSV Genome Structure (1) Cloning of ALSV Genome cDNA was synthesized by an ordinary method using oligo (dT) and random primers using RNA extracted from purified virus as a template, and cloned in E. coli. As a result, a clone containing almost the entire length of RNA1 and RNA2 was obtained. (2) Analysis of base sequence of ALSV genome The base sequence of cDNA was analyzed using dideoxy method. The sequence of the 5 ′ end region of the genome was determined after synthesis of cDNA using the 5′-RACE method. As a result, it was revealed that RNA1 was composed of 6815 bases and RNA2 was composed of 3384 bases except for poly (A) at the 3 ′ end.
These base sequences are described in DDBJ AB030940 (RNA1) and AB030941 (RNA2).
(3) When the protein coding region (ORF) in the nucleotide sequence analyzed for the protein encoded by the ALSV genome was searched, RNA1 starts with the AUG at the base number 6815 and begins with ORF1 and the base encoding the 23 KDa protein. There was an ORF2 (encoding a 235 KDa protein) beginning with AUG number 388 and almost full length. In the 235 KDa protein, conserved sequences of protease cofactor, NTP-binding helicase, protease and RNA polymerase were observed from the N-terminal side. These genome organization was consistent with the genome organization of viruses belonging to the Comoviridae family.
RNA2 encoded a polyprotein with a molecular weight of 108 KDa beginning with the 312th AUG. On the N-terminal side of this protein, a protein presumed to be an intercellular transfer protein (MP) was encoded.
(4) ALSV coat protein coding region
Using the Edman method, the N-terminal amino acid sequences of three CPs, Vp25, Vp24 and Vp20, separated by SDS-PAGE were determined. As a result, the N-terminal 15 amino acid sequences of these three types of CP are the 377-391st (Vp25), 770-784th (Vp20), and 594-608th (Vp24) positions on the 108 KDa protein encoded by RNA2. Matched the sequence. From the above results, the ALSV coat protein is present on the C-terminal side of the 108 KDa polyprotein encoded by RNA2, and the protease cleavage sites are QG (MP / Vp25), QG (Vp25 / Vp20) and EG (Vp20 / Vp24) was revealed.
[0014]
Taxonomic affiliation of ALSV Based on the physicochemical properties and genome organization of ALSV as described above, ALSV was considered to be a virus belonging to the family Comoviridae. Currently, there are three genera of the Comoviridae family: Comovirus, Nepovirus, and Favavirus. ALSV particles differ from these three genera viruses in that they are composed of three types of CP. Furthermore, in the phylogenetic analysis based on the amino acid sequence of the polymerase region of the 235 KDa protein encoded by RNA1, ALSV is expressed as above 3 It became clear that it formed a cluster different from the genus. From the above results, it was concluded that ALSV is classified into the Comoviridae family, but unlike any existing genus of viruses, it is necessary to establish a new genus in the future.
[0015]
Example 2
Application of apple small spherical latent virus (ALSV) to a vector Apple small spherical latent virus (ALSV) is a small spherical virus having a diameter of about 25 nm that infects apple without causing disease. The genome of this virus consists of two types of single-stranded RNA (RNA1, 6815 bases; RNA2, 3384 bases), RNA1 encodes a 235 kilodalton (K) polyprotein, and RNA2 encodes a 108K polyprotein ( FIG. 1).
The purpose of this subject is to use ALSV as a viral vector for easy gene transfer into fruit trees such as apples. Therefore, 1) construction of infectious cDNA of ALSV genome, 2) modification of infectious cDNA clone into viral vector, 3) introduction of foreign gene into modified vector and expression in plant as follows It was.
[0016]
1. Construction of infectious clones of ALSV genome (pEALSR1 and pEALSR2) Infectious clones of ALSV RNA1 and RNA2 were constructed as follows.
RNA was extracted from the purified virus, and cDNAs for RNA1 and RNA2 were synthesized by reverse transcriptase. Subsequently, double-stranded DNA was synthesized using RNase H and DNA polymerase and cloned with an E. coli plasmid. The 5 ′ and 3 ′ ends of the viral genomic RNA were synthesized by PCR using primers synthesized based on the base sequence and ExTaq DNA polymerase (TaKaRa). These DNAs were accurately ligated downstream of the 35S promoter (El235S, distributed by Dr. Yuko Ohashi, former Biotechnology Ministry of Agriculture and Fisheries) to construct a full-length cDNA clone for RNA1 (pEALSR1) and a full-length cDNA clone for RNA2 (pEALSR2) ( Figure 2). There will be 102 polyA sequences at the 3 ′ end of RNA1 transcribed from pEALSR1, and 65 polyA sequences at the 3 ′ end of RNA2 transcribed from pEALSR2.
Escherichia coli (DH5α) was transformed with the thus prepared exogenous gene transfer vectors of the present invention, pEALSR1 and pEALSR2, and cultured in large quantities. Both plasmid DNAs were extracted using a plasmid extraction kit from E. coli (Qiagen), suspended in Tris-EDTA (TE) buffer so that the DNA concentration was 2 μg / μl, and used for the following inoculation experiments. It was.
Chenopodium quinoa was used for the infectivity test of the constructed clone. Four leaves of C. quinoa fully developed in a glass greenhouse were inoculated with 10 μl of pEALSR1 and pEALSR2 mixed solution (final concentration of both 0.5 μg / μl) for each leaf by the carborundum method. As a result, from 11 to 12 days after the inoculation, green spots appeared on the upper leaf, and then became mosaic. This symptom was the same as that in the plant inoculated with the virus. After crushing the infected leaves, when C. quinoa was again inoculated with sap, 4-5 days later, chlorotic spots appeared on the inoculated leaves, and then mosaics appeared on the upper leaves. In addition, when Western blot analysis was performed using ALSV antiserum (obtained from the Apple Department of the Fruit and Tree Experiment Station, MAFF), three types of coat proteins of ALSV were detected (FIG. 3). From the above results, it was found that the constructed pEALSR1 and pEALSR2 have infectivity.
In order to investigate the relationship between the plasmid DNA concentration and the infection rate, the pEALSR1 and pEALSR2 concentrations were diluted in 5 stages (0.5 μg / μl, 0.2 μg / μl, 0.1 μg / μl, 0.04 μg / μl, 0.02 μg / μl). Inoculated C.quinoa. As a result, infection was observed at a concentration of 0.04 μg / μl or more (FIG. 3).
[0017]
2. The modified ALSV-RNA2 of the infectious cDNA clone (pEALSR2) encodes one type of 108K polyprotein, including 42K cell-translocation protein (MP) and three types of coat from the N-terminal side. Proteins (Vp25, Vp20, Vp24) are included. This 108K polyprotein is cleaved by the protease encoded by RNA1 after translation to yield each protein. As a first step in the modification of the vector, a plasmid (pEALSR2XB) containing two types of restriction enzyme sites (XhoI-SmaI-BamHI) repeated twice between the protease cleavage sequence (Q / G) of MP of pEALSR2 and Vp25 Built. By introducing the restriction enzyme site, the foreign gene transfer vector of the present invention, which can easily link the foreign gene, was obtained (FIG. 4).
When pEALSR2XB was mixed with pEALSR1 (final concentration 1 μg / μl) and inoculated into 5 individuals of C. quinoa, symptoms appeared in all upper C. quinoa lobes after 9-12 days. Total RNA was extracted from this infected leaf by the phenol method, RT-PCR of the full length of genomic RNA 2 was performed, and the product was treated with BamHI and XhoI. The RT-PCR product of wild-type (pEALSR2) infected leaves is not cleaved by these enzymes, whereas the product of pEALSR2XB-infected leaves is cleaved, so that pEALSR2XB is infectious and has grown Was found to have a restriction enzyme cleavage site (XhoI-SmaI-BamHI) (FIG. 5).
[0018]
3. Expression of exogenous gene (GFP) in plant using modified vector As shown in FIG. 6, a clone (pEALSR2GFP) in which a green fluorescent protein (GFP) gene was introduced into the XhoI and BamHI cleavage sites of pEALSR2XB was produced. Using pEGFP-1 (CLONTECH) as a template, a + strand primer XhoGFP (+) [5′-CC CTCGAG ATGGTGAGCAAGGGCGAGGA-3 ′] containing a restriction enzyme cleavage sequence and a − strand primer BamHGFP (−) [5′-CG GGATCC CTTGTACAGCT The GFP gene was amplified by KOD-plus DNA polymerase (TOYOBO) using CGTCCA-3 ′] (underline indicates a restriction enzyme cleavage sequence). This was treated with XhoI and BamHI and then ligated with pEALSR2XB cleaved with the same restriction enzymes. The obtained plasmid was designated as pEALSR2GFP.
pEALSR2GFP and pEALSR1 were mixed (each 0.5 μg / μl) to prepare a foreign gene transfer vector composition of the present invention and inoculated into C. quinoa. When the upper leaf one week later was observed with a stereoscopic fluorescence microscope, GFP fluorescence was observed in a portion of the vein of the inoculated leaf (FIG. 6). Further, on the 9th day after the inoculation, the propagated virus was transferred to the upper leaf of the directly inoculated leaf, and GFP fluorescence was observed along the vein in the entire upper leaf (FIG. 7). After crushing these infected leaves and inoculating C. quinoa with juice, a large amount of ring-like fluorescence was observed in the seed leaves. RNA was extracted from infected leaves by the phenol method, and then the entire length of genomic RNA 2 was amplified with reverse transcriptase M-MLV reverse transcriptase (TOYOBO) and ExTaq polymerase. The PCR product was treated with BamHI and XhoI, and electrophoretic analysis of 1% agarose confirmed the presence of the GFP gene fragment (FIG. 8).
From this, it was confirmed that the viral vector containing the GFP gene constructed in this study replicates in the inoculated leaf of C. quinoa and moves to the upper leaf, and at the same time expresses the foreign gene (GFP) in the plant body.
[0019]
【The invention's effect】
According to the present invention, introduction of a foreign gene into fruit trees (for example, apples), which is a very difficult technique, and expression of the foreign gene in the plant body can be performed easily and efficiently.
In addition, the method of the present invention is a new technique that can be used to make a virus vector that infects apples and the like, introduce foreign genes into fruit trees, realize transformation, and improve varieties or produce new varieties.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the genome structure of apple small spherical latent virus (ALSV) (top) and electron micrographs (about 250,000 times) of apple small spherical latent virus (bottom).
FIG. 2 shows the construction of a full-length cDNA clone for RNA1 (pEALSR1) and a clone for RNA2 (pEALSR2), which are infectious cDNA clones of the present invention, and a restriction enzyme map.
FIG. 3 shows the results of Western blot analysis using ALSV antiserum showing infectivity in Chenopodium quinoa by mechanical inoculation using a foreign gene transfer vector of the present invention containing pEALSR1pEALSR2 (bottom). . The relationship between plasmid DNA concentration and infection rate is shown (top).
FIG. 4 shows the modification of an infectious cDNA clone (pEALSR2), which is a foreign gene transfer vector of the present invention.
FIG. 5 shows that the exogenous gene transfer vector of the present invention, pEALSR2XB, is infectious and that the propagated virus genome has a restriction enzyme cleavage site (XhoI-SmaI-BamHI). It is the schematic diagram and the photograph of electrophoresis which are shown.
FIG. 6 shows the result of inoculating a mixture of pEALSR2GFP and pEALSR1, which is a foreign gene transfer vector composition of the present invention, into C. quinoa and observing it with a stereoscopic fluorescence microscope one week later. It is a photograph which shows a mode that the fluorescence of GFP was observed by the vein (about 250,000 times).
FIG. 7 shows the result of inoculating a mixture of pEALSR2GFP and pEALSR1, which is a foreign gene introduction vector composition of the present invention, into C. quinoa and observing it with a stereoscopic fluorescence microscope 9 days later. It is a photograph which shows a mode that the fluorescence of GFP was observed along the leaf vein in the whole upper leaf which is not (about 250,000 times).
FIG. 8 shows RNA extracted from infected leaves by a mixture of pEALSR2GFP and pEALSR1, which is a foreign gene transfer vector composition of the present invention, by the phenol method, and then amplified by PCR using reverse transcriptase. The product is treated with BamHI and XhoI, and after electrophoretic analysis of 1% agarose, it is a schematic diagram and a photograph showing that the presence of the GFP gene fragment was confirmed.

Claims (22)

  A foreign gene transfer vector composition for fruit trees comprising a vector comprising DNA for the full length of genomic RNA 1 of apple globules latent virus and a vector comprising DNA for the full length of genomic RNA 2 of apple globules latent virus.   The foreign gene transfer vector composition according to claim 1, wherein the DNA for the full length of the genomic RNA of the apple small spherical latent virus is cDNA.   The foreign gene transfer vector composition according to any one of claims 1 to 2, comprising DNA for the full length of the genomic RNA of the apple small spherical latent virus.   The foreign gene transfer vector composition according to any one of claims 1 to 3, comprising a region encoding a poly A sequence.   The foreign gene transfer vector composition according to any one of claims 1 to 4, comprising a transcriptional regulatory factor upstream of DNA relative to genomic RNA.   The foreign gene transfer vector composition according to claim 5, wherein the transcriptional regulatory factor comprises a promoter.   The foreign gene transfer vector composition according to claim 6, wherein the promoter is a plant virus-derived promoter.   The exogenous gene transfer vector composition according to claim 7, wherein the promoter is a 35S promoter.   The foreign gene transfer vector composition according to any one of claims 1 to 8, wherein a restriction enzyme site is newly introduced.   The foreign gene introduction vector composition according to any one of claims 1 to 9, wherein a restriction enzyme site is introduced at a position corresponding to a boundary between base sequences encoding two proteins in genomic RNA2.   The exogenous gene transfer vector composition according to claim 10, comprising a restriction enzyme site between the base sequence encoding the intercellular transfer protein (MP) and the base sequence encoding the coat protein (Vp25).   The exogenous gene transfer vector composition according to claim 10 or 11, wherein a base sequence encoding a protease cleavage site in a polyprotein in genomic RNA2 is repeated, and a restriction enzyme site is included therebetween.   The foreign gene transfer vector composition according to any one of claims 9 to 12, comprising a plurality of restriction enzyme sites.   An exogenous gene introduction vector composition for fruit trees comprising the exogenous gene introduction vector composition according to any one of claims 9 to 12, wherein an exogenous gene is introduced into a restriction enzyme site.   An exogenous gene transfer vector composition for apple, comprising the exogenous gene transfer vector composition according to any one of claims 9 to 13, wherein a foreign gene is introduced into a restriction enzyme site.   A method for introducing a foreign gene into fruit trees comprising inoculating fruit trees with the foreign gene introduction vector composition according to claim 14 or 15.   The explant to the fruit tree according to claim 16, which comprises inoculating the obtained juice after grinding the leaves of the fruit tree infected with the exogenous gene transfer vector composition according to claim 14 or 15. Gene transfer method.   The method for introducing a foreign gene according to claim 16 or 17, wherein the fruit tree is an apple.   A method for inoculating a fruit tree with the foreign gene introduction vector composition according to any one of claims 1 to 15, introducing the foreign gene into the fruit tree, and expressing the foreign gene in the fruit tree.   After crushing the leaves of fruit trees infected with the foreign gene transfer vector composition according to any one of claims 1 to 15, inoculating the obtained juice, introducing the foreign genes into the fruit trees, A method of expressing a gene in fruit trees.   21. The method for introducing a foreign gene according to claim 19 or 20, wherein the site where the foreign gene is expressed is different from the site of inoculation.   The method for introducing a foreign gene according to any one of claims 19 to 21, wherein the fruit tree is an apple.

Images