TW416989B - Satellite RNA associated with bamboo mosaic potexvirus and the development of satellite based vector for expression of foreign gene - Google Patents

Abstract

The invention uses the satellite RNA associated with bamboo mosaic virus(BaMV) from infected Bambusa vulgaris McClure as a material for cloning and sequencing. Nucleotide sequence revealed that this satellite RNA contains a 836-nucleotide genome and encodes a 20 KDa protein. Infectious transcripts have been generated from full-length cDNA downstream T7 RNA polymerase promoter. Precise replacement of open reading frame of cDNA of satellite RNA with sequence encoding bacterial CAT (chloramphenicol acetyltransferase) resulted in high level expression of CAT in infected dicotyledon plants, Chenopodium quinoa and tobacco (Nicotiana benthamiana) in the presence of BaMV genomic RNA. Thus, this satellite system is potentially useful as a satellite-based plant expression vector.

Description

A7 B7 416989 V. Description of the invention (1) (Please read the notes on the back before filling this page) The Central Standards Bureau of the Ministry of Economic Affairs, the Consumers Cooperative, printed the use of iransgenic plants to express foreign genes, which are current plants Commonly used methods in the world, however, whether the plant is transfected, whether it is infected with Agrobacterium tumefaciens, or treated with particle bombardment or electroporation, it requires a regeneration system that depends on plant tissue culture. It is not only time-consuming and labor-intensive In addition, a large number of hoofs must be selected under the action of a suitable promoter and observed to the offspring to obtain stable transgenic plants. Therefore, a better choice is to use a modified plant virus as a vector. The plant virus infects plants and has the characteristics of automatic replication and systemic transfer in plant cells. Moreover, the virus concentration is high, and it can be used in a short time. Can express a large number of foreign gene products in plants. In fact > Although using plant virus as a vector, despite the above advantages, the modification of the virus gene often affects its replication ability; or after the virus is replicated for several generations, the foreign gene is lost by the recombination of the virus gene . Early development of cauliflower mosaic virus as a DNA vector ’was abandoned because the capacity and recombination problems could not be overcome. In recent years, the understanding of RNA viruses and the construction of full-length infectious cDNA have greatly increased the feasibility of using leg viruses as vectors. Tobacco mosaic virus) is used as a carrier to express antihypertensive peptides in the festival (Biotechnology 11: 930-932, 1993) »while the development of plant virus satellite RNA (vector) is used as a vector, only ― Example: Masuda tax report (G Patent Publication, JP 2053491, 1990), using satellite nucleic acid of cucumber mosaic virus (CMV) as a vector to insert potato virus Y (potato vims V, PVY) 3 ' For non-standard paper, the Chinese National Standard (CNS) Λ4 is present (210X297 mm) A7 B7 416989 V. Description of the invention (2) 120 bp cMA in the translation area, so that the crops infected with the virus (p VY) Symptoms were reduced after three weeks. However, because the entire satellite nucleic acid of the courgette mosaic virus (CMV) has only 369 riboflavin, in terms of the capacity to express foreign genes, it may be of limited use. "Satellite nucleic acids are generally about 200-1500 nucleotides short. RNA molecules. These molecules do not have sequence homology with the nucleotides of viral RNA. They must be replicated in the presence of the virus gene foot A. At the same time, they are coated with diseased sheath proteins (Wcrobiol _ Rev. 56: 265-279, 1992) »At present, according to their molecular weight, they can be roughly divided into two types: the first type is a satellite nucleic acid with a small molecule, only about 30 (more than 3 nuclear fenamic acids, although it has extremely Strong PNA double-stranded structure, but does not have the ability to synthesize proteins, such as the satellite nucleic acid of cucurbit mosaic virus (CM V), so the ability to insert foreign gene fragments is limited. The second type is a large satellite with large molecular weight. Has the ability to synthesize satellite proteins, but such satellite proteins are required for its replication (Biochimie 75: 561-567, 1993), so it cannot be deleted and replaced by foreign genes. S ·· · Bamboo infected with virus Known Mosaic virus (BaMV) is a potato mosaic virus (potexvirus) with a single-stranded positive-stranded RNA genome of about 6.6kb (Phytopathology 82:73, 734 > 1992). Recently We isolated different BaMV isolates from different bamboo species, and found that 'Thai Mangosteen Isolated BaMV-V' contains not only 6,4 kb genomic RNA, but also small RNA molecules. This RNA molecule is suitable for the North paper scale. China National Standard (CNS) A4 specification (2 〖0X297mm) 4 1 ^ 1 I— I 1 ^ 1 In 1 y ^-I (Please read the precautions on the back before filling this page) -5 " Ministry of Economic Affairs China National Standards Bureau®; Printed by the Industrial and Consumer Cooperation Department of the Ministry of Economic Affairs

418989 S V. Description of the invention (3) Fangzao, nucleoside sequence analysis, and other experiments prove that this short RNA molecule is the satellite nucleic acid of BaMV, and this satellite nucleic acid is not only the potato X virus group (potexvirus guoup) The first found in) is also the only satellite nucleic acid with a rod-shaped appearance in plant viruses, and this satellite nucleic acid can carry foreign DNA fragments, and foreign genes are used in this satellite nucleic acid vector (satellite-RNA based vector) in BaliV In the presence of genomic RNA, a large number of foreign gene products can be rapidly and rapidly expressed in plants. SUMMARY OF THE INVENTION The present invention includes a satellite nucleic acid obtained from a bamboo mangosteen strain infected with bamboo mosaic virus, in addition to preparing the satellite nucleic acid to contain a full-length cDNA, and its in vitro transcript is biologically active. This satellite nucleic acid has developed into a plant vector that can carry foreign genes. The content of the present invention is described as follows: 1. Material acquisition: The present invention is to harvest the leaves of BambuS'a vuigaris McClure from the bamboo cultivation area of the Taipei Botanical Garden and purify the virus. RNA extraction and agarose gel electrophoresis to isolate RNA, it was found that this mangosteen mosaic virus (BaP-V) isolate not only contains a genomic RNA of about 6400 nucleotides (called the genomic R_NA as LRNA), but also Contains an RNA molecule of about 850 nucleotides (this RNA molecule is called sBaMV). The paper ruler is applicable to the Chinese national standard ((: that) 8 4 specifications (210 father 297 mm) 5 (please read the matter on the back of Jing before filling in the clothes page)

416989 V. Description of the invention (4) In order to understand the characteristics of sBaKV of Taishan bamboo streak virus isolates, the northern heterozygous method was used to evaluate the relationship between the genomic RNA of bamboo mosaic virus and sBaMV, and the RA probe of the virus was found. (L-cDNA) does not hybridize to sBaHV. Conversely, it uses sBaMV-specific probes (S-cDNA), nor can it hybridize with BaMV-V genomic RNA, showing the lack of cross-hybridization between viral RNA and heterologous probes. BaHV There is no required sequence homology between the genomic RNA and sBaMV. 2. Prove that sBaMV in BaMV-V isolates has the characteristics of satellite nuclear Jun: In order to understand the characteristics of sBaMV in BaitV-V isolates, this nucleic acid was separated from the non-denaturing low-solubility agar colloid after electrophoresis and carried out Inoculation experiment ° Whether inoculated with Chenopodiom quinoa or barley protoplasts, it was confirmed that this sBaifV alone cannot cause infection when it is present. In addition, this sBaMV RNA was placed in a test tube containing rabbit reticulocyte lysate and analyzed for in vitro transfer products. It was found that sBaMV can effectively synthesize polysaccharides with a molecular weight of about 25 kDa, and immunoprecipitation experiments The reaction showed that the protein synthesized by sBaMV did not react with BaKV virus, sphingomyelin. The seal of the staff consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). From the above analysis of northern hybridization, inoculation experiments, and analysis of in vitro translation products, sBaMV has the characteristics of satellite nucleic acids. And it must be in the presence of the BaliV genome R'NA in order to replicate. 3. sBaMV was verified as a newly recorded RNA molecule by analysis of cDNA nuclear | acid sequence: sBaMV KNA separated by electrophoresis was used as a template, and double-stranded cDNA was synthesized according to the Riboclone cDNA Synthesis System (Promega). Chinese National Standard {CNS) Ad specification (2I0X297 mm) 416989 A7 B7_____ V. Description of the invention (5) PUC119 plastid for nucleotide sequence analysis. The results are shown in Figure 3. Excluding the residues of the polyfluorene tail, the full length of sBaMV contains 836 nucleotides, including 25.5% A, 29.3% C, 24.8% G, and 20.49ί U base combinations & The nucleotide sequence was compared with GenBank and found that it does not have homology with any known gene, virus or satellite nucleic acid, so the satellite nucleic acid does have novelty, and the nucleotide sequence number given by GenBank is: L22 It's 62. 4. Biologically active sBaMV in vitro transcripts can be synthesized in a test tube: sBaMV KNA purified using colloids as a template, oligonucleotide 5 · -GTCGACTC TAGA (T) 15) as a primer, and the first strand of cDNA was synthesized to contain Ί7 polymerase promoter oligonucleoside BS19 (see Example 5), a second strand of cDNA was synthesized and then cloned into PUC119 plastids. After selection, pBSF4 is the strain containing the full-length sBaM cDNA (Figure 8). After linearization of plastid PBSF4 by Xbal, in vitro transcription reaction is performed. The synthesized transcript is mixed with the genomic RNA of BaMV-V and inoculated with barley protoplasts or plant resveratrol. Mutations can be modified at the DNA level in pBSR plastids. 5. The sBaMV translation region can be deleted without affecting its replication: In order to understand the relationship between the sBaHV protein translation region and sBaMV replication, this full-length biologically active satellite nucleic acid cDNA, pBSF4 (Figure 8), has been mutated ( Figure 6 A): For example, pBSF5, change the satellite protein start code 160 AUG to I60UUG; pBSF6, after the start code 160 AUGG, insert a nucleic acid C to cause a frame shift; pBSF7 * at the 450th This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm > 7 (Please read the notes on the back before filling this page) Printed by the Employees' Cooperative of the Prospective Bureau of the Ministry of Economic Affairs _ _ n I n- ------nl--I ~ ^-n-order -1 ^ ---- I n-n--nl-I n Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 4189S9_B7__ V. Invention Note (6) After nucleotides, 38 nuclear fens. Acids were deleted; pBSF8, which substantially deleted the region between the 280th and 559 sarcosinates; PBSF9, completely removed the translation region of satellite proteins, leaving only satellites 5 'and & ends of nuclear translation non-translated region-transcripts of the above mutants in BaKV gene In the presence of RNA, whether inoculated with barley protoplasts or plant reeds, it was shown that these in vitro transcripts did not lose their ability to replicate nucleic acids. Only the replication capacity of BSF9 was reduced in protoplast experiments, about 1 of the original BSF4. 5¾, so it is proved that satellite proteins are not required for satellite nucleic acid replication and can be deleted. 6. Foreign genes can be expressed in plants by vectors containing satellite nucleic acids: In order to explore the ability of satellite nucleic acids to carry foreign genes, the reporter gene C'A T (Chloramphenicol acetyl transferase) replaces the translation region of SBaMV to obtain a recombinant heteroligand pBSCAT containing the CAT gene (Figure 7A). After in vitro hybrid transcript BSCAT is synthesized, it is used to inoculate barley protoplasts together with BaMV gene RNA. It was found that the replication ability of BSCAT transcripts was similar to that of BSF4, and it was not affected by the insertion of foreign genes. Its replication ability was not affected. Using ELISA kit (Boehringer Mannheim GmbH), the content of CAT enzymes in infected leaves was immunodetected It shows that one gram of resveratrol leaves can produce 25 ng of CA 7 protein, which proves that SBaMV has the following characteristics: Ability of foreign gene protein * Therefore, this satellite nucleic acid has the potential to be developed as a vector for plants to express foreign genes. Based on the above results, the present invention has the following characteristics: (1) This satellite nucleic acid vector is in the presence of viral genome RNA According to the paper size, the Chinese National Standard (CNS) A4 regulations apply (230X297 meters) 8 (please read the precautions on the back before filling this page)

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 416989 V. Description of Invention (7) A large number of foreign gene products can be replicated and accumulated in infected plants within 6-14 days, which is more than the current establishment of transgenic plants. It takes months or hours to express foreign genes, which is not only fast, time-saving, labor-saving, but also can achieve the effect of radon concentration. (2) In the presence of viral genome RNA, this satellite nucleic acid vector can not only infect monocotyledonous plants, such as the original host plant bamboo, barley, corn, etc., but also dicotyledonous plants, such as reeds, millennia red and tobacco (Nicotiana benthamiana) The dicotyledonous plant Xuecao C Nicotiana benthamiana) is a very suitable host plant. When the vector and the viral genomic RNA coexist, the viral genomic RNA can systematically infect the plant, and it is a disease-free infection. Without any agronomic traits, foreign products can be produced in large quantities in tobacco plants. (3) Compared with other developed plant RNA viruses as vectors, this satellite vector has many advantages: Except that the concentration of satellite nucleic acid is usually higher than that of viral gene RNA, foreign genes are generally embedded in the gene of virus' R NA However, the present invention embeds a foreign gene into the translation region of a satellite nucleic acid. Therefore, RNA recombination or RNA instability due to foreign gene insertion will be greatly reduced. At the same time, compared with the developed satellite nucleic acid vectors, the satellite fumaric acid of the present invention is confined to large satellite nucleic acids, and therefore has a larger space than small satellite nucleic acids, and can be embedded with a larger amount of molecular molecules. This paper size applies to Chinese national standards (CNS > Α4 size {210X297 mm > 9 (Please read the precautions on the back before filling this page)

4169S9 A7 B7 V. Description of the invention (8) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (4) The satellite nucleic acid vector of the present invention, in addition to the successful expression of foreign genes such as the bacterial reporter gene (CAT) in this description, The laboratory has also successfully expressed the 3A mobile protein gene of CMV. Therefore, any potential foreign gene, such as ribozyme containing BaHV sheath protein-specific sequences, has the potential to develop BaMv disease; as well as surface antigens of Heptis virus B, can be used Plants produce medical vaccines; and any other enzymes, polypeptides, or proteins that can change the amino acid content of crops with medical effects, may be expressed in the plant by this satellite carrier. That is, one of the objects of the present invention is to isolate and confirm a novel satellite nuclear warfare, and determine the entire nuclear fenamic acid sequence. The second object of the present invention is to synthesize a biologically active satellite nucleic acid cDNA plastid. In the presence of BaKV genomic RNA, its in vitro transcript can infect plants. *. · The third purpose of the present invention is to provide satellite nucleic acid plastids that can rapidly express foreign genes in plants (Figure 8). The translational region of the essential body can be replaced by foreign genes, and the in vitro transcripts are present in the BaMV gene body RNA. In addition, foreign gene products can be rapidly expressed in infected plants. The above-mentioned objects, features and advantages of the present invention will be understood from the examples and drawings disclosed in the following description of the invention. These descriptions are for illustration only and not for limitation. The scope of the present invention is defined by the scope of patent application. (Please read the notes on the back before filling in the last page)

This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210x297 public epidemic) 10 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 4169S9 —, __ · — · + 11 ~ V. Description of the invention (9) Example 1 Satellite nucleic acid Separation, extraction, and northern blot analysis were performed. The leaves of Bambusa vulgaris McClure were harvested from the bamboo specimen park of the Taipei Botanical Garden. Using Lin, NS and others published in Phytopathology Vol. 81, No. 155 in 1991. The method described on page 55 was used to purify the Taimosa mosaic virus (BaMV-V) virus. RNA was extracted at the same time, and the RNA was isolated by electrophoresis. The electrophoresis was performed with 1% agaric colloid in Tris-borate buffer solution (Tris-borate.plO), and the known characteristics of green bamboo mosaic virus isolate (BaMV —〇) RNA (Phytopathology 82: 73, 734, 1992) 'do the same treatment' as a control. The results are shown in Figure (1 A). The virus from the green bamboo mosaic virus berry isolate (BaMV-0) contains only one genomic RNA (called L KNA) of approximately 6,4 nucleotides (line 2). ); However, the virus of Bacillus thuringiensis isolate (BaMV-V) additionally contains an RNA molecule of about 850 nucleotides, called sBaMV (line 3), which has a molecular weight greater than that of squash mosaic virus (CMV) Satellite Nucleic Acid (Line 1) ° To clarify the characteristics of sBaMV of a Thai mosaic bamboo mosaic virus isolate 'Evaluate the bamboo mosaic virus genome by northern hybridization method * ?!' | 4 and sBaMV Relationship, Figure: 1B shows that the virus's genomic probe (L_cDNA) can be isolated from the genomic RNA of green bamboo mosaic virus isolate (BaMV-0) (line 2) and Taishan bamboo mosaic virus The size of the paper body of the strain (BaMV ~ V) applies to the Chinese National Standard (CNS) A4 specification (210x297 mm > 11 』Γ.) ≫ ^ 11 ^ 1 u ^ i ft ^ ^ fi ^^^ 1 — ii — ^ ϋ ^^^ 1 —1 ^^^ 1-- (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 4169S9 B7 Five 'Invention Note (10) RNA (Line 3), and there was no mismatch with sBaMV (line 3) of Squash mosaic virus (CMV), or its satellite nucleic acid (line 1) or Taisho bamboo mosaic virus isolate. However, using the sBaHV specific probe (S-cDNA), in contrast, it can hybridize with Taisho bamboo mosaic virus sBaMV nucleic acid (line 3), but cannot hybridize with other virus KNAs (line 1, 2). The lack of cross-matching between the virus's RNA and heterologous probes shows that there is no significant sequence homology between the genomic RNA of bamboo mosaic virus (BaMV) and sBaKV. Example 2 Isolation and inoculation experiment of sBaMV nucleic acid To understand the characteristics of sBatfV of Taishan Bamboo Mosaic Virus isolate, the nucleic acid of this virus was subjected to non-denaturing 1¾ low-solubility agar gel colloid electrophoresis. Two sections of KNA generated by electrophoresis, Genomic RNA and sBaifV_ RNA were cut out from electrophoretic colloids, and then separated by phenol extraction and alcohol precipitation. The purified RNA was re-dissolved in sterile distilled water, quantified by UV absorption spectrum, and stored at -70 ° C for inoculation test. The Taishanbamboo streak virus gene RNA isolated by this method * does not contain sBaMV RNA, which is called BaMV-V / S-RNA, and the isolated sBaMV does not contain bamboo mosaic virus gene body RNA. Both are inoculated Before, all were tested by nucleic acid probe hybridization test and confirmed that they were not contaminated with each other '> a. Plant inoculation experiment Dilute the isolated 1 sBaMV RNA in 100 μ 1 inoculation buffer solution (0.05 Μ Κ2 ΡΡ (Μ, 0.1M) separately Glycine, 100 μ g / ml The paper size is applicable to China National Standard (CNS) 8 4 specifications (2IOX29? Mm) I ------------- Order (please first Read the notes on the back and fill in this page) 4X6989 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (11) Bentonite, pH 9.3, inoculated with the leaves of the plant Chenopodium quinoa, After one to two weeks of observation of the inoculated leaves, neither local lesions were found nor any amount of sBaMV RNA could be recovered. However, the same concentration of green bamboo mosaic virus (BaMV-0) gene body β NA or BaMV-V / S- genomic RNA for the same inoculation analysis can be performed on inoculated leaves It produced about ⑽ local lesions, so it proved that sBaMV could not cause infection when it was alone. B. The protoplast inoculation experiment selected the young leaves of Hordeum vulgare L. cv. Larker which grew for 7 days as the isolated mesophyll. The main sources of plastids, the steps related to the isolation of protoplasts, the inoculation of viral RNA, the extraction of protoplast RNA, and northern hybridization, are all according to the inventor's 1992 published in Bot. Bull. Acad. Sin. Vol. 33 No. 271- 275 and Phtopathology Vol. 82, 731-34. Barley protoplasts were inoculated with viral RNA, and 24 hours after inoculation, all nucleic acids of the protoplasts were extracted, and the analysis of genomic RNA and sBaMV was performed by northern heterozygous analysis. Cumulative placement, each test has 3 pieces of transfer paper, mixed with L-, S-and two kinds of mixed CDNA probes, respectively, and all can get the same results. 囵 2 shows that inoculation with BaMV-VRNA Barley protoplasts, after 24 hours, mixed cDNA probes can detect gene remuneration and sBaMV RNA replicas (Figure 2 A, 2B, 2C, line 1). Applicable paper size China 囡Furniture standard (CNS > A4 size (210X297 mm) I) (Please read the precautions on the back before filling this page)

4169S9 V. Description of the invention (12) Using the L-cDNA probe alone, full-length genomic RNA (6.4 Kb) and two major sub-genomic RNAs with a length of 2.0 Kb and 1.0 Kb can be observed (Figure 2 B , Line 1.) With the S-cDNA probe, haploids of sBaMV ENA with tritium concentration and a slow-moving diploid were detected (Figure 2C, line 1). When BaMV-V / S-isolated RNA was first verified by sBaP RNA contamination using ink dot hybridization method, inoculation of protoplasts * could only detect genomic LRNA and two subgenomic RNAs (Figure 2) , Line 2), when using the S-cDNA probe alone, no heterozygous signal was shown (Figure 2C, line 2). The sBaMV RNA itself is printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page). The assessment of whether it has the ability to replicate is to inoculate it separately into protoplasts, and the analysis of northern heterozygotes does not matter There was no miscellaneous signal of that probe (Figures 2A, 2B, 2C, line 3), so it proved that sBaMV RNA itself would not replicate. However, when the protoplasts were inoculated with a mixture of BaMV genomic RNA and sBaMV RNA, the genomic RNA and rain subunits could be observed. The replication signals of phylogenetic and sBaMV RNA (Figure 2 A, 2 B, 2C, line 4) . The protoplasts inoculated with steamed water instead of virus'RNA were not presented with any miscellaneous signals (Figure 2A, 2B, 2'C, line 5), and the sixth line in Figure 2A, 2B, 2C Marked by purified BaP-V β NA. Example 3 sBaMV cDNA Synthesis, Colonization and Nucleotide Sequencing Using sBaMV f? NA isolated from agar colloid cleavage as a template, using oligonucleotide (d T) 15-Xbal as a primer, and using the medulla of birds Cellular disease disease paper size General Chinese National Standard (CNS) A4 specification (210X 297 gong) 14 A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 416939 B7 V. Description of the invention (13) Avian myeloblasto'sis virus ) The first cDNA is synthesized by reverse transcriptase. The reaction conditions for the synthesis of the first and second strands of cDNA were synthesized with reference to the Kiboclone cDNA Synthesis System (Promega). After the double stranded cDNA fragments were synthesized, they were cut with Xbal and then ligated to pUC119 plastid cut with Smal and Xbal. It was transformed into E.coli Γ »Η5α and screened for Southern heterozygosity with S-cDNA probe. The plastids containing the sBaMV specific gene were cloned. The p-somatic pBSNL2 was selected using the S-cDNA probe. It contains 822 sBaifV nucleotide c brain inserts and 17 poly (A) residues. Therefore, the Dideoxynucleotide chain termination method was used. (Proc. Natl. Acad. Sci. USA 74: 5463-". 5467, 1977). The 839 nucleotides of the double-stranded cDNA were sequenced in two directions. This PBSNL2 plastid, although containing 822 nuclei of sBaMV ^: Acid cDNA, but lacks 14 nuclear fenamic acids corresponding to the 5 'end of sBaMV. Among them, the 5' end sequence directly isolates sBaMV RNA from the agar-agar colloid. As a slab, directly uses B S-8 (3 ' TCTTTGTGATCT GGT 5 ') is a primer, which is complementary to the 36th to 50th nucleotides and performs direct RNA sequencing at the 5' end of sBaMV RNA (Anal. Bi.ochem. 157: 275-282, 1986). As shown in Figure 3, after removing the poly (A) tail residues, the full-length sBaMV RNA contains 836 ribofenic acids, including 25.5% A, 29.3% C, 24.8% G, and 2G. 4% U salt base. GenBank analysis and comparison found no homology with any known gene, virus or satellite nucleic acid, so the satellite nucleic acid does have a new The paper size is suitable for China National Standards (CNS) A4 (210X297 Gongchu) 1 5 (Please read the note ^^ on the back before filling this page)

416939 V. Description of the invention (14) The nucleotide sequence number is: L22762 < The six nucleotides before the nucleotide sequence are GAAAAC, which are the same as the six nucleotides before the BaMV genome RNA, and the two are KNA In the meantime, there are some similar sequences scattered in the 5 'end non-translated interval. The gene body of sBaMV has a large open transliteration interval. The first start code AUG starts after 159 non-coding nucleotides and ends with 709 nucleotides with UGA terminal base code. This open translation interval can encode a polypeptide with a molecular weight of 20,154. The polypeptide contains 183 amino acids, and the 3 'non-translated interval of sBaMV RNA has a polyadenylation signal of AAUAAA (Figure 3) Example 4 In-tube translation and immunoprecipitation reaction The in-tube translation reaction is to prepare RNA as described above. The RNA sample is placed in a test tube containing rabbit reticulocyte lysate (RRL, Promega). The immunoprecipitation system of the translation product is based on C anti-BaMV-cp) containing anti-bamboo mosaic virus sheath protein. The translation products and immunodepleted deposits were analyzed by electrophoresis (? AG) 'using polyacrylamide colloids with a linear gradient of 8-2G sodium dodecylsulfuron sodium, and detected by fluorescence X-ray photography (Huorography) (Eur. J 'Biochem .46: 83-88, 1974) Consumption cooperation between employees of the Central Bureau of Standards of the Ministry of Economic Affairs___Printed by the company ---- ^-(Please read the precautions on the back first (Fill in this page) As shown in Figure 4, BaMV-VR Ν Λ and BaMV-〇RNA, both

Both large molecular weight peptides with a molecular weight of 160 kDa can be synthesized (line U 2), but BaMV-V R N A can also synthesize a molecular peptide with a molecular weight of approximately 25 kDa (line 2). Purification and separation of sBaMV fi NA from agarella colloidal paper. Standard Chinese Standard (CNS) No. 84 (2 丨 〇 > < 297g *) Shellfish consumer cooperation cooperation printed by the Central Bureau of Standards of the Ministry of Economic Affairs 416989 V. Description of the invention (15), also shows that it can effectively synthesize multiple peptides with a molecular weight of 25 kDa (line 3), and the translation product of BaMV-V / S- genomic RNA and BaMV-VRNA Comparison of the products (line 1), lack of 25 proteins (4), it can be seen that the sBaHV RNA can encode a 25kDa protein, which is compared with nucleotide sequence analysis. It is predicted that the ability to encode a 20kDa protein is slightly out of line. The isoelectric point of the protein product was too high (PI = ^ .25) ', which caused the deviation of the swimming speed in the colloid. Immunoprecipitation test results showed that all translation products of BaMV-VRNA and BaHV-0 RNA were unable to react with the spleen virus of the bamboo mosaic virus selling protein, showing that the protein synthesized by sBaHV and the sphingomyelin protein of BaMV had no blood.淸 reaction. From the above Northern Heterozygosity Analysis, Vaccination Experiment 'Nucleic Acid Sequence Analysis, and Analysis of In Vitro Translation Products, it was proved that sBaMV is not a BaMV subgenomic RNA or a defective interferring RNA molecule, but a BaKV satellite Nucleic acids can only be duplicated in the presence of BaMV genomic RNA, and this satellite nucleic acid has the ability to synthesize a 20 kDa protein, which is different from commonly known small satellite nucleic acids that do not contain a significant translation region. Example 5 Construction of a full-length infectious sBaMV cDNA single strain

Using colloidal purified sBaMV RNA as a template, oligo nucleic acid GTCG This paper uses Chinese National Standard (CNS) A4 specifications (2 丨 .0X297 mm) (Please read the precautions on the back before filling this page)

41B9S9 A7 B7 V. Description of the invention (16) (Please read the precautions on the back before filling in this page) ACTCTAGA (T) 15) as a primer, the first cDNA was synthesized by the above method, and BS19 (5 '-TCCCTGCAGTMTACGACTCACTATAGAAAACTCACCGCAACGA) is The second primer (containing the T7RNA polymerase promoter) uses T4DNA polymerase to synthesize a second strand of cDNA (liol. Cell. Biol. 4: 2876-2882, 1 9 8 4). The full-length cDNA was separated by low melting point dogwood colloid electrophoresis, cut with PstI and Xbal, then adhered to pUC119 plastid cut by PstI and Xbal, and then transformed into DH5fr E. coli. After breeding, PBSF4 contains a full-length sBaMV cDNA strain (see Figure 8).

After plastid PBSF4 was linearized by Xbal, extracorporeal transcription reaction was performed (J. Virol. 61: 1457-1465, 1987). The transfectants synthesized from 0.6 ug of plastid DNA were mixed with log BaMV-V / S-R N A in 200 ui of distilled water and inoculated with quinoa leaves. Seven days after inoculation, all RNA was extracted from the leaves (Nucleic Acids Res. 17: 2362, 1989), and the northern hybridization was performed. As shown in FIG. 5, both L- and S- mixed cDNA probes could detect high levels of RNA. The concentration of genomic RNA and sBattV RNA (line 3) proves that the sBaHV transcript synthesized in vitro and in the presence of BaMV genomic RNA also has the ability to replicate. Example 6 Analysis of Mutants of Satellite Nucleic Acids and Their Activity Analysis Printed by the Consumers 'Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 11 In order to understand the relationship between satellite nucleic acid protein translation regions and satellite nucleic acid replication, the full-length biologically active satellite nucleic acid cDNA' pBSF4 is referred to Kunkel Methods (Methods Enzymol. 154: 367-382, 1987). Several mutations were made. The paper size is applicable to Chinese national standards (Liyang > 8 4 specifications (210 fathers 297 mm). 18 Ministry of Economic Affairs and Central Standards Bureau employees. Cooperative Du printed A7 __ 416989_B7___. __ V. Description of the invention (17) (Figure 6a): (1) PBSF5: Change the starting code of the satellite protein 160AUG to 160UIJG, then there is 205 nucleotides downstream of it Another start code, so it has the ability to synthesize 18 kDa protein ° (2) pBSF6: After the start code of 160 AUGG, insert a nuclear fenamic acid C to cause a transcoding shift (frame shift). It is only expected to synthesize changed satellites Protein 6 kDa (3) pBSF7: after the 45G nucleotide, if 38 nuclear nuclei are deleted, its open translation interval (◦ RF) is shortened in advance to kDa and only 11 kDa altered protein can be synthesized. (4 ) pBSF8: Large If the region between the 28th and the 559th nucleotides is deleted, only a 10 kDa red star protein is synthesized. (5) PBSF9: the translation region completely removing the satellite protein 'only the non-translated region of the satellite nucleic acid is retained 5'and; T-terminal " The mutant transcript was synthesized by the method described above, and the v / s gene ㈣..NI 荏 —in ... X 丄 .1 basket Taili—1 health_set. Body … '-..............- Take all the RNA lines; square hybrids. Figure 6 B and C show that these in vitro transcripts are Without losing the replication ability of its nucleic acid, the transcripts BSF5 ~ F8 have an average replication ability of about 20-55% of BSF4 ', but the replication ability of Road Four is about 1% of the original BSF4. (Figure 6C), it may be After the translation region was deleted, the structure of the RNA was changed due to the inoculation test of the leaves of Tribulus terrestris. The results were similar to those of the protoplasts. Only BSF9 could not replicate in the leaves (Figure 6A). However, it was proved that the satellite protein 'This paper rule is not applicable to the Chinese family standard (CNS) A4 specification (210X297 public envy) {Please read the precautions on the back before filling in this tile)

416989 V. Description of the invention (18) Satellite nucleic acid replication can be deleted.寊 Example 7 Preparation of satellite nucleic acid recombinant heteroligand In order to investigate the ability of satellite nucleic acid to carry foreign genes, the translation region of sBaMV was replaced by the reporter gene CAT (Chlora [叩 henicoi acetyl transferase)). CAT gene from pCM7 (Promega, Wisconsin), using two primers BSCAT1 (5 '-ΤΑ TCCAAGACGATGGAGAAAAAATC-3') and BSCAT2 C 5,-CAGCCTCT GGGAGGTTACGCCCCGCCCTG-3 ') for polymerase chain reaction (PCR) ) Reaction 30 cycles > Each cycle contains 941 '45 seconds; 55 ° C, 45 seconds; 2 ° C, 丨 minutes' After copying the DNA fragment, it was' adhered to BstXI and BstXI after cutting by BstXI and EcoNI EcoRI-cleaved PBSF4 vector was selected to obtain the recombinant heteroligand pBSCAT containing the CAT gene (Figure 7A). Printed by the staff of the Central Bureau of Standards of the Ministry of Economic Affairs. Printed by the company (please read the notes on the back before filling out this page). Using the aforementioned method, synthesize the in vitro heterozygous transcript BSCAT * BaMV-V / S-gene RNA. Inoculate reed leaves. Seven days after the inoculation, the virus was purified and the viral RNA was extracted. The figure shows that the cumulative child of BSCAT transcript is similar to SSF4, which proves that BSCAT transcript is not affected by the insertion of foreign genes and affects its replication ability. It also proves that this recombinant heterozygous transcription Body, coated with a virus sheath protein. To test the ability of recombinant hybrid transcripts to express the CAT gene * BaMV-V / S-genomic RNA and BSCAT transcripts are co-inoculated with quinoa plants. Paper size is applicable to China National Standards (CNS) A4 specifications (210X297 male) A7 B7 416989 1 'Explanation of the invention (19). After 6-7 days of inoculation, the leaves are ground with 4 times the volume of buffer (0.5 M borate buffer, 1 mM 巳 DTA, 0.5% 2-mercaptoethanol' pH 9.0), and then The layer of gauze was passed through the furnace. The filtrate was added with 2% Triton X-100 ′ and stirred for 0 minutes, and centrifuged at 3000 rpra ’to remove plant residues. Two times more methanol containing 0.1 MNH4C1 was added. After thorough mixing, it was placed at -70 t: for 2 hours. After centrifugation, the precipitate was suspended in distilled water, and the suspension was immunoassayed with CAT ELISA k! T (Boehnnger Mannheim GmbH) to detect the CA T enzyme content.妁 Figure.7C. One gram of resveratrol leaves can produce 25nS CAT protein, which proves that BSCAT has the ability to express foreign gene proteins. 5 (Please read Back 1 & Precautions Ear Hard. Quot

、 1T Printed by I Industry Consumer Cooperatives

W 2 One centimeter 416989 Employees of the Central Bureau of Standards of the Ministry of Economic Affairs and cooperation Du printed A7 B7 V. Brief description of the invention (20) Figure 1 Electrophoresis diagram (A) of virus RNA and analysis of northern mismatches (B , C). (1) Purified cucurbit mosaic virus (CMV) RNA. (2) Purified green bamboo mosaic virus (BaMV-0) R N A. (3) Purified mangosteen mosaic virus (BaMV-V) R N A. (A) Electrophoresis map of viral RNA in a non-denaturing 1% agar colloid. Electrophoresis was performed with Tn s-Junjun salt buffer solution for 1: ethtdium bromide staining, and (B, C) viral RNA was separated during electrophoresis. After that, it was transferred to the hybrid membrane of H-Bond and hybridized with L-cDNA probe (B) and S-cDNA probe (C), respectively. L-cDNA and S-cDNA probes were prepared using L RNA or sBaMV RNA as templates, respectively. In the presence of avian myeloblastosis virus (Promega, Madison, WI), Six nucleic acids or 32P-labeled cDNA synthesized by oligod (T) 15 primers. Figure 2 Northern Heterozygous Analysis of the Replication of R N A in Barley Protoplasts. Protoplasts were inoculated separately (1) BaMV-V R N A. (2) BaMV-WS- R N A. (3) sBaMV R N A. (4) Mixture of BaMV-V / S- and sBaMV R N A. (5) Distilled water. (6) The purified BaMV-V RNA is a molecular marker. This paper size applies the Chinese National Standard (CNS) Λ4 now (210X297 cm) 22 (Please read the precautions on the back before filling .: '; This page)

Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Consumers Cooperatives 416939 Λ7 ---- ^ __ B7-’— V. Description of the Invention (21) (A) Detection by L- and S-mixed cDNA probes. (B) L-cHntA probe detection. (C) Detection of S-cDNA probes l-cdna and s-dm probes are prepared as shown in Figure 1. 3 The nucleotide sequence of the full-length sBaMV of Taishan bamboo mosaic virus isolate and its amino acid sequence i. 4 Viral RNA was analyzed in rabbit fragment red 51 ball lysate (RRL) 's translation product' in a S-20% linear gradient sodium dodecane sodium polypropanolamine colloid (SDS-polyacryl £ mide) electrophoresis system for analysis ( 1) BaMV-0 RNA, (2) BaMV-V RNA = (3) sBaMV purified from the mucosa „(4) BaMV-v / S- RNA. (5) distilled water. The size of the molecular weight marker is marked first. On the left side of the line, kDa is the leading position.

Fig. 5 Biological activity of Northern Heterozygous analysis of nucleic acid full-length CDNA pBSF4 transcripts in inoculated Chenopodiuro quinoa} leaves. Inoculation of reed leaves (1) BaMV-V / S-RNA (2) BaMV 'VRNA (3 ) BaMV-V / S-RNA and pBSF4 mixture of in vitro transcripts, 7 days after inoculation, to extract all rNA, this paper size applies Chinese National Standard Xin (CNS) Λ4 specification (210: < 297 mm) ( Please read the precautions below and fill in this page). Ordered by the Central Bureau of Standards of the Ministry of Economic Affairs and printed by 51.416989. Description of invention (22) Northern hybridization, detection with L- and S-cDNA mixed probes Assays, the preparation of L- and S-cDNA probes is shown in Figure (1), and the * arrow in the figure indicates the position of sBaMV. Figure 6 Bamboo full-length virus cDNA (pBSF4) and its mutant Atlas and infectivity test (A) Gene map, mutant construction and infectivity analysis of pBSF4 and its mutants pBSF5 to F9. &Quot; + "represents infectious; 1 represents incapable of infection. (E, C) Northern Miscellaneous Test the in vitro transcripts of pBSF4 ~ F9 in the presence of BaMV RNA and inoculate barley Infectivity of protoplasts. Barley protoplasts were inoculated with BaMV- V / S- RNA and (1) pBSF4 (2) pBSF5. (.., 3) pBSF6 (4) pBSF7 (5) pBSF8 (6) PBSF9 transcripts. The mixture or (7) was inoculated with only PBSF9 transcripts. After 24 hours, the entire RNA was extracted and separated by L-cDNA probe (B) and s cDNA probe (C), and L- and S-cDNA probes were detected. The preparation is shown in Figure 1 'described.' _ _ Figure 7 Bamboo mosquito virus satellite nucleic acid full-length cDNA (pBSF4) and a recombinant hybrid containing the bacterial chloramphenicol, ketamine, and acetaminophen transfer enzyme (CAT) gene ( pBSCAT) Gene Map of Transcript (BSCAT) 'Infectivity and Biological Activity Assay (A) Gene Map of pBSF4 and pBSCAT. This paper scale applies to China National Standard (CNS) Λ4 specification (2 丨 〇297297) 4 (Read the precautions on the back before filling this page)

416989 at B7 ~ »-I I ----- m V. Description of the Invention (23) (B) Northern Miscellaneous Test pBSF4 and pBSCAT infectivity test. Resin leaves were inoculated with a mixture of BaMV-V / S-RNA and (I) PBSF4 and (2) pBSCAT transcripts. Seven days after inoculation, the virus was purified, and viral RNA was extracted with (a) L-cDNA probe and (b ) S-cDNA probes are northern heterozygous. (C) In the same inoculation test as in Figure (B), the leaves of Tribulus terrestris are 'extracted from all plant proteins' 7 days after inoculation, and the content of chloramphenicol, chloramine, phenylene, and acetic acid (CAT) in the inoculated leaves is tested by £ USA . (3) The CAT enzyme with a concentration of 诏 has been used as a concentration control group. Figure 8 蓂 pBSF4) map containing the bamboo mosaic virus satellite nucleic acid gene. 'Foreign genes can be inserted between the BstXI and Econi restrictions. (Please read the cautions on the back before filling in this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs This paper size applies to China's national standard (CNS> M specification U! 〇 X 4 7 public funds) 25

Claims (1)

Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs of the People's Republic of China 416989 6. Application for Patent Scope 1. A method for rapidly expressing foreign genes in plants 'uses bamboo mosaic virus satellite nucleic acid as a vector' and performs the following steps: a. Preparation of full-length infectious bamboo mosaic virus satellite nucleic acid cDNA: that is, a synthetic satellite nucleic acid full-length cDNA (contains 836 nucleotides whose poiy (A) at the 3 'end is not counted), and T7 is added at its 5' front end. The promoter is cloned in the PUCI19 vector (derived from pUC 19, Clontech Laboratories, Inc. Palo Alto, California 94303, USA), called pBSF4, (Figure 8), and its in vitro transcript has Biological activity, can 'replicate in infected plants in the presence of BaMV-assisted viral gene RNA; b. Using satellite nucleic acid as a vector to express the function of foreign genes' will translate the above-mentioned satellite nucleic acid-containing cDNA translation region (nucleotide 丨(60-70S) deletion, incorporation of human foreign gene constructs between Bs tXI and EcoNI restriction enzymes to obtain satellite nucleic acid recombinant hybrids (Figure 8), and the use of in vitro transcription systems to prepare exogenous Satellite nucleic acid recombinant transcripts are inoculated in the presence of BaMV helper virus genomic RNA. Foreign genes satellite nucleic acid recombinant transcripts are biologically active, and foreign genes perform their functions in infected plants; c. Satellite nucleic acid as a carrier * Production of foreign gene products in plants: Embed foreign genes in the satellite nucleic acid vectors described above to generate an in vitro transcription system to prepare satellite hybrid nucleic acid recombinant transcripts containing foreign genes. In the presence of BaMV gene RNA, inoculate plants. Foreign genes can be quickly And a large number of them are expressed in plants, and leaves can be harvested 6-14 days after inoculation to purify foreign gene products. This paper uses Chinese National Standards (CNS > M size (210X297 mm)) (Please read the precautions on the back before filling this page) ABCD 416989 VI. Patent Application Fan Garden 2. If the scope of the patent application is the first item, it contains satellite nucleic acids of all isolates of bamboo mosaic virus. 3. As claimed in item 2 of the patent application scope, which includes all PBSF4 derived protoplasts. 4. The method according to item 2 of the scope of patent application, wherein the planting line is selected. Any dicotyledonous plant is selected. 5. If the method according to item 2 of the patent application scope, the soil-growth plant line selects any of the dicotyledons. I Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs This paper size is applicable to China National Standards (CNS) Λ4 grid (210X297 mm)