CN1231587C - Beet black withered virus as expression carrier of foreigh gene - Google Patents

Abstract

The present invention describes a whole nucleotide sequence of a beet black scorch virus (BBSV). Under the control of eukaryotic promotors and prokaryotic promotors, the cDNA clone in a whole BBSV genome can be transcribed. The viral RNA which is generated has infection capability to host plants. When the situation that the viral RNA is inserted into a medusa green fluorescent protein gene (GFP) is taken as an example, the example indicates that the obtained recombination BBSV can be used as a carrier to carry genes of which the source is not viruses and express the gene in plants.

Description

Sugar beet black scorch virus as expression vector of exogenous gene

1. Technical field

The invention relates to a carrier used in genetic engineering. Specifically, it is a virus-derived vector that can carry foreign genes and express them in plants.

2. Background technology

Since the 1980s, a kind of black scorch beet virus disease has been found in my country's main sugar beet producing areas such as Xinjiang, Ningxia, Inner Mongolia, Gansu, Heilongjiang, and Jilin, which has brought huge losses to production [Cui Xingming et al., Shihezinong Journal of the Academy, 10(1), 73-78(1988)]. Symptoms of the disease on sugar beets are mainly as follows: leaves stand upright, black scorch spots appear between leaf veins, leaf margins curl inward, and root hairs are necrotic, etc. The damage to sugar beets has surpassed that of beet necrotic yellow vein virus BNYVV), and increasingly serious [Liu Jiexian et al., Chinese sugar beet, (3), 30-31 (1995)]. In 1989, the diseased area in Ningxia alone accounted for 69.2% of the sugar beet production areas in the whole region, of which 4.6% had lost production or lost use value. After more than 10 years of research, the inventor proved for the first time in the world that the pathogenic microorganism that caused the disease was a new virus, and named it as Beet black scorch virus (BBSV). In-depth and comprehensive research has been carried out on serology, particle morphology and its mode of transmission, and a series of research results have been obtained.

It has been proved that BBSV is a spherical virus with a diameter of 28nm, and the coat protein subunit is a single component with a molecular weight of about 24.5kDa. Under natural conditions, it only infects beets, but under artificial mechanical inoculation conditions, it can infect 13 species of plants in 4 families, such as amaranth, apricot and tobacco. BBSV is carried by Olpidium brassicae through zoospore flagella in vitro, and has only a weak serological relationship with tobacco necrosis virus (Tobacco necrotic virus, TNV) willow isolates [Jiang Junxi et al. , Journal of Jiangxi Agricultural University, 21(4), 525-528(1999)].

Beet black scorch virus is a new virus found only in my country, and there is no similar report about the disease in other countries or regions in the world. China is the only country that has carried out research on BBSV, but current research on the virus is limited to basic biology such as virus biology, morphology, and serology. The present invention describes the nucleotide sequence and structural features of the BBSV genome for the first time, and confirms the functions of different genes on the genome. On this basis, the virus genome is further modified to make it capable of carrying foreign genes and being highly efficient in plants. Expressed viral vectors.

3. Contents of the invention

1. Determined the whole genome nucleotide sequence (SEQ ID NO 1) of this new virus of BBSV, and determined the size and structure of the BBSV genome.

Sequence analysis showed that the BBSV genome was a single-stranded positive-sense ss(+) RNA consisting of 3641 nucleotides and containing 6 open reading frames (ORFs). The ORF located at the 5' end of the RNA encodes a 22kDa protein P22, which can produce an 82kDa read-through protein P82 after the amber stop codon (Reading-through, R/T) of the protein, which is a viral RNA-dependent RNA polymerase. The ORF at the 3' end of BBSV RNA encodes a 24.5 kDa coat protein (CP). There are three small ORFs between the polymerase gene and the coat protein gene, encoding the 4.2kDa protein P4.2 and two 7kDa proteins P7a and P7b, respectively (see Figure 1). According to GenBank search and comparison, although BBSV has the highest homology with tobacco necrosis virus strain D (TNV-D), the sequence identity is only 61%. Based on the research results of BBSV biological and serological manifestations, fungal transmission characteristics, genome size and structure, nucleotide sequence homology, and satellite RNA components present in individual isolates, the inventors named BBSV tobacco A new virus of the genus Necrovirus.

2. The BBSV infective cDNA clone pUBF52 under the control of the T7 promoter was constructed, which contains the nucleotide sequence of SEQ ID NO 1 and the T7 promoter, and the BBSV genome can be transcribed under the control of the T7 promoter to regenerate Active BBSV genomic RNA.

3. Constructed the BBSV invasive cDNA clone pRBS2 under the control of the CaMV 35S promoter, which contains the nucleotide sequence of SEQ IDNO 1 and the CaMV 35S promoter, through the effect of the 35 promoter, the cDNA of BBSV can be obtained in plants Transcription, regenerates BBSV genomic RNA with infectious activity.

4. Utilizing the obtained BBSV invasive cDNA clones pUBF52 and pRBS2, different regions of the virus genome were mutated and transformed, the functions of different genes in the BBSV genome were confirmed, and their functional regions were located.

5. On the basis of precise positioning of the BBSV genome functional genes, carry out deletion mutation or replacement mutation [the present invention replaces the coat protein gene of BBSV with the jellyfish green fluorescent protein (GFP) gene], Under the premise of ensuring that the virus's infective ability and its own replication ability are not significantly affected, the pathogenicity of the virus is weakened, and on this basis, a virus expression vector capable of carrying foreign genes is constructed.

6. Infect and transform host plants, and measure the effect of using BBSV as a foreign gene expression vector.

Transforming BBSV, a new virus that is only found in my country so far, into a virus vector that can carry foreign genes, is easy to operate, and has high infection efficiency. It is used to use plants to produce medicinal proteins. It is original in materials .

4. Description of drawings

Figure 1. The structure of the BBSV genome and the size of the encoded protein

Figure 2. PCR amplification results of BBSV full-length cDNA

A is the λDNA/EcoR I+Hind III double enzyme digestion Marker;

B is the PCR amplification product of the full-length cDNA of BBSV 3.64kb

Fig. 3 In vitro transcription product of BBSV invasive cDNA clone pUBF52 under the control of T7 promoter

A is the in vitro transcript of pUBF52

B is BBSV RNA

Figure 4 Northern blot detection of in vitro transcript infection activity of BBSV infective cDNA clones under the control of T7 promoter, where g is BBSV genome; sg1 is subgenome 1; sg2 is subgenome 2

A is the in vitro transcript inoculation of BBSV invasive cDNA clone pUBF52 under the control of T7 promoter

B is blank control

C is inoculated with BBSV RNA

Figure 5 Western blot detection of in vitro transcript infection activity of BBSV infective cDNA clones under the control of T7 promoter

A is blank control

B is inoculated with BBSV RNA

C is inoculated with in vitro transcripts of BBSV-infectious cDNA clone pUBF52 under the control of T7 promoter

Figure 6: Northern blot detection of the infection activity of BBSV invasive cDNA clones under the control of 35S promoter

1 is blank control

2 Inoculation of in vitro transcripts of the BBSV full-length invasive cDNA clone pUBF52 under the control of the T7 promoter

3 for inoculation with BBSV RNA

4 and 5 were inoculated with the BBSV invasive cDNA clone pUBS2 under the control of the 35S promoter

Figure 7 Schematic diagram of the structure of four deletion mutants (pBDCP, pDCPn100, pDCPc6 and pDCP18) in the BBSV genome

Figure 8 Northern blot detection of in vitro transcript infection activity of different deletion mutants of BBSV genome

A: blank control

B: The inoculum is RNA of BBSV

C: The inoculum is the in vitro transcript of BBSV full-length cDNA clone

D-G: The inoculum was the in vitro transcripts of BBSV coat protein gene deletion mutants (pBDCP, pDCPn100, pDCPc6 and pDCP18), respectively

Figure 9 observes the expression of the jellyfish green fluorescent protein (GFP) gene in the BBSV virus vector with a laser confocal microscope (Confocal), and green is the expressed jellyfish green fluorescent protein

A is the leaf of Amaranthus quinoa inoculated with the in vitro transcript of pUBF52 (containing BBSV full-length cDNA, without inserting GFP gene)

B is pUB-GFP (controlled by T7 promoter) in vitro transcript inoculated leaves of Amaranthus quinoa

C is the leaf of Amaranthus quinoa inoculated with pRBS-GFP (controlled by 35S promoter)

5. Specific implementation

Example 1: Cloning and sequence analysis of BBSV whole genome cDNA

Isolate the virus from sugar beet showing black scorch symptoms in Ningxia, Xinjiang, Inner Mongolia, Gansu, Heilongjiang, Jilin and other main sugar beet producing areas, and inoculate host plants such as Chenopodium amaranticolor and Tetragonia expansa by rubbing, and use PEG precipitation The virus was extracted from the diseased leaves of Chenopodium amaranth by conventional methods such as centrifugation and differential centrifugation, and then extracted with phenol-chloroform to obtain the RNA of BBSV. According to the results of agarose gel electrophoresis, the BBSV nucleic acid is ssRNA with a size of about 3.6-3.7kb. The elution experiment of Oligo(dT) cellulose column proves that there is no Poly(A) tail sequence at the 3′ end of BBSV RNA.

According to the standard operating procedure of GIBCO BRL, the RNA of BBSV Ningxia isolate was first polyA-tailed. Using Oligo(dT)12-18 as a primer, cDNA synthesis was performed on the tailed viral RNA using the cDNA synthesis kit from Promega Company. The resulting double-stranded cDNA was blunt-ended with T4 DNA Polymerase and then cloned into the SmaI site of pGEM-7Z(+). T4Polynucleotide kinase was used to end-label the viral RNA with γ- ³² p-ATP, and dot hybridization with the recombinant plasmid, and 4 positive clones were screened --- pGB212, pGB159, pGB327 and pGB146. Sanger's dideoxy termination method was used for sequence determination with ABI 377 DNA sequencer, and the three positive clones pGB212, pGB159 and pGB327 had the same 3' end. According to the measured sequence, new primers were designed and synthesized, and the synthesis of cDNA was continued, and the nearly full-length genome of BBSV was finally obtained. The 5' and 3' end sequences of the BBSV genome were obtained and confirmed using Invitrogen's 5'RACE and 3'RACE kits, respectively, so as to obtain the full-length sequence of the genome.

According to the basic molecular biology experiment method described in the "Molecular Cloning Experiment Guide", primers BB-18 and BB-14 were designed according to the end sequence of BBSV genomic RNA, and the purified BBSV RNA was used as a template to carry out reverse transcription-chain polymerase Reaction (RT-PCR, denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 4 min), a 3641bp full-length cDNA of BBSV can be obtained (see Figure 1).

The sequence of primer BB-18 is: 5′- TGTAATACGACTCACTATAG AAGAAACCTAACCAGTTCTCGTTGATCAGCGAT-3′, corresponding to 1-34nt of SEQ ID NO 1, and the underline is the introduced T7 RNA polymerase promoter sequence.

The sequence of primer BB-14 is: 5′-TCC CCCGGG CCACCTGGAAGACCAGGTATAT-3′, complementary to 3618-3641nt of SEQ ID NO 1, the underline is the introduced Sma I site

The various test methods and test techniques involved in this example are common knowledge, and those of ordinary skill in the art can implement the basic molecular biology test methods described in the "Molecular Cloning Experiment Guide".

Example 2: Construction of BBSV infective cDNA clones under the control of T7 promoter, in vitro transcription and infective activity assay

Primers BB-18 and BB-14 were designed according to the BBSV RNA terminal sequence, wherein the 5' end of the BB-18 primer introduced the T7 RNA polymerase promoter sequence, and the 5' end of the BB-14 primer introduced the Sma I restriction site (in BBSV There is no such site in the whole sequence). Using the purified BBSV RNA as a template, RT-PCR amplification (denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 4 min) yielded a full-length cDNA of 3.64kb BBSV (Figure 2). After the PCR product was cut flat by T4 DNA polymerase, it was ligated into the SmaI site of the vector pUC18. After screening and identification, the recombinant plasmid pUBF52 was obtained.

After the recombinant plasmid pUBF52 was linearized with endonuclease Sma I, about 100-200ng of linearized DNA was used as a template, and T7 RNA polymerase was used to transcribe in vitro, and a transcript with the same size as BBSV viral RNA could be transcribed (Fig. 3 ). After inoculating the leaves of Amaranthus quinoa by rubbing the in vitro transcripts, 3-4 days later, the same small scabs as those inoculated with BBSV RNA can be produced, and there is no significant difference in the severity of the disease, indicating that the BBSV genome can be transcribed under the control of the T7 promoter. Regeneration of BBSV genomic RNA with infectious activity.

Example 3: Northern blot detection of in vitro transcript infection activity of BBSV invasive cDNA clones under the control of T7 promoter

The in vitro transcripts of pUBF52 and viral RNA were used to inoculate the leaves of Amaranthus quinoa respectively, and the total RNA of the leaves was extracted, separated by 1% denatured agarose gel electrophoresis, and then transferred to Hybond-H ⁺ nylon membrane by capillary method via 20xSSC. According to the basic experimental method described in the "Molecular Cloning Experiment Guide", using the 0.3kb non-coding region at the 3' end of BBSV labeled with digoxin as a probe, pre-hybridization (pre-hybridization at 42°C for 5-6 hours), hybridization (42 ℃ hybridization for more than 12 hours), membrane washing and color development and other steps for Northern blot detection, the results showed that in addition to the main band of BBSV genomic RNA (3641bp in size), there were two small subgenome bands (Figure 4). It shows that the in vitro transcript of pUBF52, like BBSV RNA, can not only infect the host plant, but also replicate in the host.

Example 4: Western blot detection of in vitro transcript infection activity of BBSV invasive cDNA clones under the control of T7 promoter

Use pUBF52 in vitro transcripts and viral RNA to inoculate leaves of Amaranthus quinoa respectively, take 0.5-1.0 grams of diseased leaves after 3-4 days, grind them in liquid nitrogen, add 300 μl of protein loading buffer (40mM Tris-Cl, pH6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.1% bromophenol blue), shake and boil in water bath for 10 minutes, immediately place on ice to cool, centrifuge to get the supernatant, separate by 12.5% SDS-PAGE gel , the protein was transferred to nitrocellulose membrane (NC membrane) by electrotransfer method, and the BBSV-specific antiserum made by the inventor and goat anti-rabbit IgG labeled with alkaline phosphatase were used as the first antibody and the second antibody respectively. The secondary antibody, using NBT and BCIP as chromogenic substrates, was used to detect the expression products of pUBF52 in vitro transcripts and viral RNA in host plants by Western blot. A specific band consistent with the size of BBSV coat protein (24.5kDa) was found (Figure 5), indicating that the in vitro transcript of pUBF52 can not only infect the host plant, but also replicate and produce functional proteins (such as coat protein) in the host.

Example 5: Northern blot detection of BBSV invasive cDNA clone infection activity under the control of 35S promoter

According to the nucleotide sequence of SEQ ID NO 1, primers BB-26 and BF-6 were designed and synthesized, and pUBF52 was used as a template for pCR amplification (denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 3 min, and a total of 30 cycle) to obtain the full-length cDNA of 3641bp BBSV, which was blunt-ended with T4DNA Polymerase and then cloned into the Bal I site of the expression vector pRT103 containing the 35S promoter to obtain the recombinant expression plasmid pRBS2. Plasmid pRBS2 under the control of 35S promoter was directly rubbed to inoculate the leaves of Amaranthus chinensis, while the in vitro transcripts of pUBF52 (containing T7 promoter) and BBSV RNA were used to inoculate the leaves of Amaranthus chinensis respectively as a control. After 4-5 days, total RNA was extracted from leaves. After electrophoresis, the RNA was transferred to a nylon membrane. According to the basic experimental method described in the digoxin kit, pre-hybridization (pre-hybridization at 42°C for 0.5 hours), hybridization (hybridization at 42°C 4 hours), membrane washing and color development and other steps for Northern blot detection, the results showed that the BBSV invasive cDNA clones under the control of the 35S promoter were the same as the BBSV invasive cDNA clones under the control of the T7 promoter, and both were hostile to the BBSV host The plant Amaranthus quinoa is invasive, indicating that the invasive cDNA clone under the control of the 35S promoter can not only infect the host plant, but also replicate in the host (Figure 6).

The sequence of primer BF-6 is: 5'-GGGCACCTGGAAGACCAGGTA-3', and is complementary to 3641-3618nt of SEQ ID NO 1

The sequence of primer BB-26 is: 5'-AAGAAACCTAACCAGTTTCTCG-3', and corresponds to 1-22nt of SEQ ID NO 1

Embodiment 6: Northern blot detection of BBSV genome mutant infection activity

According to the basic molecular biology experimental method described in the "Molecular Cloning Experiment Guide", the coat protein gene (located at the 2644-3342 base of the nucleotide sequence of SEQ ID NO 1) located at the 3' end of the BBSV genome was completely or partially cloned After deletion, four deletion mutants of the BBSV genome --- pBDCP, pDCPn100, pDCPc6 and pDCP18 were obtained (Fig. 7). The invasive cDNA clones of the four deletion mutants were constructed in the same way as the BBSV whole-genome invasive cDNA clones. After in vitro transcription by T7 RNA polymerase, the in vitro transcripts were inoculated into Chenopodium amaranth, and the total RNA from the inoculated leaves was extracted 3-4 days later, and Northern blot was performed using the α- ^32P -labeled BBSV 3′-terminal non-coding region as a probe Detection results showed that RNA bands of corresponding sizes could be detected in the full-length invasive clone and the four deletion mutants ( FIG. 8 ). It shows that the four deletion mutants of BBSV coat protein gene can not only be transcribed under the control of T7 promoter, regenerate RNA with infection activity, but also can replicate in the host.

Embodiment 7: The constructed BBSV vector can carry the jellyfish green fluorescent protein (GFP) gene and express it in plants

Using DNA recombination technology, insert exogenous genes [taking jellyfish green fluorescent protein (GFP) gene as an example] into the constructed BBSV virus vector, and test whether the obtained recombinant BBSV vector can carry genes from sources other than viruses and reproduce in plants. in the expression.

According to the basic molecular biology experimental method described in the "Molecular Cloning Experiment Guide", according to the nucleotide sequence of SEQ ID NO 1, primer BB-20 and primer BB-21 were designed and synthesized, and pUBF52 was used as a template for reverse PCR amplification. After agarose gel electrophoresis, the amplified product of 5.9 kb was recovered, and then blunt-ended with T4 DNA polymerase. The obtained target DNA fragment included the sequence of the vector pUC18 and the gene except for the coat protein (located at the nucleotide of SEQ ID NO 1 The rest of the BBSV genome outside the 2644-3342 bases of the acid sequence). According to the nucleotide sequence of jellyfish green fluorescent protein (GFP) gene [Cormack, B.P. etc., Gene, 173, 33-38 (1996)], design and synthesize primer GFP1 and primer GFP2, obtain complete GFP gene through PCR amplification . The above 5.9kb fragment was connected with the GFP fragment to obtain the recombinant plasmid pUB-GFP controlled by the T7 promoter and carrying the jellyfish green fluorescent protein (GFP) gene, wherein the coat protein gene on the BBSV genome was replaced by the complete GFP gene.

Using the same strategy, the BBSV invasive cDNA clone pRBS2 under the control of the 35S promoter constructed in Example 5 was replaced and transformed to obtain the recombinant plasmid pRBS- GFP, in which the coat protein gene on the BBSV genome is replaced by the complete GFP gene.

The plasmid DNA of pRBS-GFP and the in vitro transcript of pUB-GFP were respectively inoculated into Chenopodium chinensis, and then Northern blot detection was performed with digoxin-labeled BBSV genome probe and GFP gene probe respectively after 3-4 days. The results showed that, Both the BBSV genome and the GFP gene in the two constructed BBSV vectors (pRBS-GFP and pUB-GFP) could be transcribed and replicated in the inoculated plants. Confocal laser scanning microscope (Confocal) scanning pUB-GFP in vitro transcripts inoculated Amaranthus quinoa leaves, can observe the existence of green fluorescent protein, indicating that the GFP gene can not only be transcribed in the constructed two BBSV virus vectors, but also can Expression follows expression of the BBSV genome (Fig. 9).

The sequence of primer BB-20 is: 5'-TTATTGACTATACTAGAAAGC-3', and is complementary to 2643-2623nt of SEQ ID NO 1

The sequence of primer BB-21 is: 5'-ATCCCACATCCTGGTGTGG-3', and corresponds to 3342-3361nt of SEQ ID NO 1

The sequence of the primer GFP1 is: 5'-ATGAGTAAAGGAGAAGAA-3', and corresponds to 1-18nt of the GFP gene

The sequence of primer GFP2 is: 5'-TTATTTGTATAGTTCATCC-3', and it is complementary to 717-698nt of GFP gene

BBSV-seq.WorkFile

Organization Applicant

----------------------

Street: No. 2, Yuanmingyuan West Road, Haidian District, Beijing

City: Beijing

State:

Country: China

PostalCode: 100094

PhoneNumber: 62892710

FaxNumber: 62892012

Email Address: bnyvv@public.bta.net.cn

<110>OrganizationName: China Agricultural University

Application Project

-------------------

<120>Title: Beet black scorch virus as an expression vector of foreign genes

<130>AppFileReference: Revision Notice--Submit the standard version of the nucleotide sequence of beet black scorch virus.

<140>CurrentAppNumber: 02155378.5

<141>CurrentFilingDate: 2002-12-11

sequence

--------

<213>OrganismName: beet black scorch virus

<400>PreSequenceString:

aagaaaccua accaguuucu cguugaucag cgaucaugga uucaaucccg uaugugaucc 60

ugcgcauacu cgauuuuauc uuccacucca ucuucuuucc aucuuuacuc uuuaucauca 120

accacaacac caccauccug ugggcaugug ccugugccua uggguucuac cgugcauucc 180

gccucaucuu caagauaaag guggaaguac accccagccac ccgagccguc uucaaggaua 240

uggucacucg cuuccagcgg gagaguaugu ucucaccuga cgacgaagug ccggagggca 300

ucccuaucca cgaggauguu gaccuuguua gcgaccccac acacaaagau auuaagaggg 360

uccgugcuag caggcgaguc ucuuaugccg ugaggguugc ccauguagcc aagucuaagg 420

ugggauugcu cgccaauacc aaggcgaaug agcuggugua cucccgucuu ugccgagacg 480

agaugguac ccacggugug cgcccaucgc acauugcaca cgcagugccg cuugcugucg 540

cggccugcuu cauaccguug gacagugauu uccuugcagc uucuauuaga aacugcgaug 600

agauggagga gcggagggcc guacuagggc ccucauagg aaaauaggga ggccuacucu 660

gcaccagcgg guuuaccacg ccuacuuggc gugguaaucc agaggguuug cuggugaaga 720

gaggaccacc ucuggccaaa ccuaggaaac uguaccguuu uucuggguuu gggacucaua 780

uacgguacgg agugcacgau cacucauugg gcaaugugcg gaagggacuu guugugcggc 840

uauucauggu ugaaaccaag gauggcuuag cuccaacacc acagcccacc cuggcgugua 900

cgccaaguua ucccgguuuc augacuguua guggccaacc uaaccucgac caccagauua 960

acauacgagc auucuucgga uuuuauucug gucgcaaauu agagagguac caacaggccg 1020

uggagucguu agcaauccgc ccaauagggg uacaggaugc ugggcuuagc acguguguua 1080

aggcugaaaa auuaaacauc ucagccaaac ccgacccggc accuaggguu auucagccua 1140

ggucgccgag guacaaugug gaaguuggac gguuccucag acacgcugag gaacaucugu 1200

ucgacgccau caaccgugug uauggugggc gaacgguauu caagggguug aaugccgauc 1260

aggcuggcau ggagaugcaa gcuauguggc aagaauucga caauccugug gguauuggua 1320

uggaugccuc ucguuucgac caacacgucu cuaaggaggc guuggaguuu gaacacaaaa 1380

ucuggcuauc cauguaccau ggggcugaca ggaaaacauu gucgaagcug uuggggaugc 1440

aaauccacaa ccgcggucuu gccagaugcc cugauggaga aaucagguac acgguuaagg 1500

ggugucguau gucuggggac aucaacacau cuucuggaaa cugcuauauc augugugcuu 1560

cggugcacaa uuauugcagc caguugggug ucaagagauu caggcuugcc aacaauggug 1620

augauugcau gcuugucguc gaagccaagg augaagcacg ugucaggcag ggacucaucg 1680

aguauuauag ggaauugggu uucaccauga aaguggaacc uacagucuau gaacucgagc 1740

acuuggaguu uugccagaca cguccagucc uugucgaugg ggcauaucga auggugcgca 1800

aucucacca gggcaugugu aaggauguuc acucuugca cgaucuuggu aguaggaaag 1860

cugcugaagc uuggguuuca gcgguugga cuggaggccg cgugaugaau gauggaguac 1920

cagugcucaa aucauucuuc augcaguuuc cccuauccuc uggaccuaaa accaagucug 1980

acaugagugu accguugcag gaagauugga aauacaaauu caaucggacu ggguguuuca 2040

agaacuuggc accccacucca caaucccgcu acucauuuug gcgugcguuu ggagugcuac 2100

cagaugaaca gauugcccug gagaaugggu uuucucgucu cagcuuugau aagcuggacc 2160

aggacaccca ggaagaaguc agccuccucc aguucucugg ggcaugaaaa ccuaaccacu 2220

uuucauggaa caacagcgga gugaacaacg ucgugaucgu agagugaaa guagaucgga 2280

ggacaggaag ucuaugucug auguagggca aucugcuguc aauagggaag cagaugucaa 2340

gaaagauaug gguccaucgg uuucuaugac gguggugggg gagaacguag aguuuacaca 2400

acauuucac uucugaaaug agcaucauuu augucguaca ggagaagccu ucuggguuuc 2460

ucguguggc auugguuguu gcaauugugu gcauuauugg acucuuaucg uacacucccac 2520

cugaaagacu uaaccacucu uaucacgaaa acaaucagaa gacgcaauac auaacuauug 2580

gaggagcauc cacuaguaaa guugccacga acugaauuuu cugcuuucua guauaguacaa 2640

uaaauggcac cuaagcgcaa uaaaggaggc aagaaguccc gcauguccga ugagacagug 2700

cgggcuccug cugcaggagg cguuguacaa cgcacaccug gcauuccucc ccgcauuagg 2760

uccaccacua uugguacgcg uggcaccaac acugagcugc ucgcuggagu gaaugucgcu 2820

gcggcggggag cuuucucagu uguuggcgcu ggucuuuucc ccagcaaccu ugguuggcuc 2880

aaugggauug cuuccaauua uagcaaauuu agauggcuug cuaucaagcu caucuacauu 2940

cccauuguuc cuaccacuac cgcuggggca augaccaugg cuuuaacgua ugauccugcu 3000

gaugcuacgc caacuaguuu ccaacaagug caacagaugu auaacagcau cacagcaccu 3060

gucugggcug gauuugaugg agcuacuguu cagcugcuag gggagagacc aacaacuggg 3120

BBSV-seq.WorkFile

gcugugugca uugaugugga uguaaaucgg uuuggauuua caugguacag guaugcuacg 3180

cuugcugcca uuaccgcacu cacugcaaau gauaggaauc uuuauauucc uaguguuggc 3240

aauguggcua cgucuggugg uacugcagcc accaauguug gcaaucugau gauaaaguac 3300

agcauugagc ucauugagcc aauaccugcu gccauuaauu agaucccaca uccuggugug 3360

guuaauccag ugaaaaauau uaggaaaucc uugggauuca caccccaggg aggauggcuu 3420

gcauagugcag guauguuga guuacacuau gagguauugg ugcugaaucc uggaaacag 3480

gcuugacagg uuuggguugu uccagaccga uguauuaccc aagauacuca ugguacugua 3540

cuagaaaaca cuaugcgugc gcacacggcu ggcuaugucc uaucauagcu gggggccccg 3600

gagugcgaaa cccucuuaua uaccuggucu uccaggugcc c 3641

<212>Type: RNA

<211>Length: 3641

SequenceName: beet black scorch virus

Sequence Description:

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: 5'UTR

<222>LocationFrom: 1

<222>LocationTo: 35

Other Information:

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF1, RNA polymerase

<222>LocationFrom: 36

<222>LocationTo: 647

Other Information:

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF2, RNA polymerase

<222>LocationFrom: 36

<222>LocationTo: 2207

Other Information: produced by translational readthrough of ORF1

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF3, 5K protein

<222>LocationFrom: 2104

<222>LocationTo: 2244

Other Information:

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF4, 7K protein A

<222>LocationFrom: 2225

<222>LocationTo: 2416

Other Information:

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF5, 7K protein B

<222>LocationFrom: 2418

<222>LocationTo: 2615

Other Information:

CDSJoin: No

Features

-------

Sequence: beet black scorch virus:

<221> FeatureKey: ORF6, coat protein

<222>LocationFrom: 2644

<222>LocationTo: 3342

Other Information:

BBSV-seq.WorkFile

CDSJoin: No

Features

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Sequence: beet black scorch virus:

<221> FeatureKey: 3'UTR

<222>LocationFrom: 3343

<222>LocationTo: 3641

Other Information:

CDSJoin: No

Journal

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Sequence: beet black scorch virus:

<301>Authors: Cao Yunhe, Cai Zhunan, Ding Qun, Li Dawei, Han Chenggui, Yu Tialin, Liu Yi

<302>Title: The complete nucleotide sequence of beet black scorch virus (BBSV), a new member of the genus Necrovirus

<303>Journal Name: Archives of Virology

<304>Volume: 147

<305> Issue: 12

<306>PageRange: 2431-2435

<307> Date: 2002-12-18

<308>DBAccessionNumber: AF452884

<309>DBEntryDate: 2002-12-30

<313> From:

<313> To:

Claims (6)

1. An isolated cDNA molecule whose nucleotide sequence is SEQ ID NO.1. 2. A plasmid vector, which contains the nucleotide sequence shown in SEQ ID NO.1 and its upstream T7 promoter. 3. The plasmid vector according to claim 2, characterized in that: Utilize DNA recombination technology, GFP gene is inserted into the position of the coding coat protein of the cDNA molecule described in claim 1 of complete or mutant transformation, and along with the right The expression of the cDNA molecule described in claim 1 is expressed, wherein the mutation transformation is the deletion or partial deletion of the coat protein gene in the cDNA molecule, and the cDNA molecule described in claim 1 is carrying the GFP gene while its own replication ability is not Not affected. 4. a plasmid vector, which contains the nucleotide sequence shown in SEQ ID NO.1 and its upstream CaMV 35S promoter. 5. The plasmid vector according to claim 4, characterized in that: utilize DNA recombination technology, the GFP gene is inserted into the position of the coding coat protein of the cDNA molecule described in claim 1 of complete or mutant transformation, and along with the right The expression of the cDNA molecule described in claim 1 is expressed, wherein the mutation transformation is the deletion or partial deletion of the coat protein gene in the cDNA molecule, and the cDNA molecule described in claim 1 is carrying the GFP gene while its own replication ability is not Not affected. 6. A method for preparing a transgenic plant, characterized in that the vector according to claim 4 and claim 5 is transferred into the plant, and the exogenous gene is expressed.

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