CN114457142A - Method for efficiently tailing and rapidly amplifying terminal sequence of plant dsRNA (double-stranded ribonucleic acid) virus genome and application of method - Google Patents

Abstract

The invention provides a method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome and application thereof. The method is characterized in that total RNA is denatured into single-stranded RNA by heating; adding A tail to the 3' end of the single-stranded RNA; using oligo (dT)₁₀-RT reverse transcribing RNA into cDNA; dividing both ends of each segment of the viral genome into a5 'end region and a 3' end region by taking cDNA as a template to perform Multiplex PCR; and (4) running the gel, recovering a target band, connecting the target band to a cloning vector, and sequencing to obtain the whole terminal sequence of the genome. The method has low requirement on RNA quality of the sample, oligo (dT)₁₀-RT with Anchor₂₄the-F primer can be commonly used for identifying the terminal sequence of the dsRNA virus genomeThe method is suitable for obtaining the terminal sequence of the dsRNA virus genome in batches, and is beneficial to obtaining the full-length sequence of the unknown dsRNA genome.

Description

Method for efficiently tailing and rapidly amplifying terminal sequence of plant dsRNA (double-stranded ribonucleic acid) virus genome and application of method

Technical Field

The invention relates to the technical field of biology, in particular to a method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome and application thereof.

Background

Among the known plant viruses, most viruses are RNA viruses. Double-stranded RNA viruses (dsRNA) belong to the class of RNA viruses and are named because their nucleic acids are complementary double-stranded RNA. Most dsRNA viral genomes are multi-segmented, and each segment contains one gene.

At present, the dsRNA purification method mainly comprises the following 3 methods: 1. the dsRNA extracted by the method is low in quality by using TRIzol reagent; 2. performing density gradient centrifugation on the extracted total RNA in cesium chloride or lithium chloride by using an ultracentrifugation method to finally obtain purified dsRNA, wherein the method is limited in popularization and application due to small amount of extracted dsRNA, high cost and complex operation; 3. The purification method using cellulose powder to adsorb dsRNA is the most commonly used method for extracting dsRNA virus, but the cellulose powder (CF-11) used for purifying the dsRNA virus at present is stopped and cannot be used normally any more, and the method for extracting high-quality dsRNA needs to be put into a sample metered by grams.

The traditional methods for identifying the terminal sequence of dsRNA virus genome are 2 types: 1. use of a segment requiring double-ended modification (5' -end PO)₄3' end NH₂) The single-stranded deoxynucleotide ' joint ' is connected to the 3' end of the dsRNA, only T4 RNA ligase can be used in the connection process, the connection efficiency of the enzyme is low, the method has higher requirement on the purity of the dsRNA to be connected, the method is not suitable for samples with only a small amount of virus materials, the time consumption is long in the process of extracting the high-quality dsRNA, the operation is complex, and the tail end of the extracted dsRNA can be degraded by partial base sequences and cannot obtain a complete dsRNA tail end sequence; 2. the method has the advantages that a SMARTer technology is used, a section of nucleotide joint is added at the 5' end of dsRNA, and then two terminal sequences of the dsRNA fragment can be identified. In both of these conventional methods, a reverse transcription primer is designed for reverse transcription for each terminal sequence identification, and the cDNA after each reverse transcription can only be identifiedA corresponding one of the terminal sequences.

Poly (A) polymerase has high tailing efficiency, 20-200A bases can be added to the 3' end of RNA, and the poly (A) polymerase does not depend on a template and can catalyze ATP to be sequentially doped into the 3' end of the RNA in an AMP form, namely, poly adenosine tails are added to the 3' end of the RNA, but the poly (A) polymerase only takes single-stranded RNA as the template.

Multiplex PCR is also called Multiplex PCR, and this method can use more than 2 pairs of primers to generate more than 2 nucleic acid fragments by PCR amplification. The Multiplex PCR is very suitable for samples with a small amount of templates, and can maximally improve the use efficiency of the templates.

Camellia latent virus 1 (CCV 1) belongs to dsRNA virus, and its genome is composed of three-segment dsRNA. Because tea contains secondary metabolites such as polysaccharide, polyphenol and the like, high-quality dsRNA is difficult to obtain according to the traditional method for purifying dsRNA, RNA with poor quality is difficult to clone to the terminal sequence of a viral genome and subsequent experiments are difficult to extract, and therefore, a new method which has general requirements on RNA quality and can efficiently amplify the terminal sequence of the plant dsRNA viral genome is needed.

Southern rice black stripe rice virus (SRBSDV) belongs to the genus Fijivirus (Fijivirus) of the Reoviridae (Reoviridae), and its genome consists of ten-segmented dsRNA. Because the number of gene segments of SRBSDV is large, the conventional molecular cloning method is time-consuming and labor-consuming to obtain the complete terminal sequence, and therefore, a new method for rapidly identifying the terminal sequence of the multiple segments is needed.

Disclosure of Invention

The invention aims to provide a method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome comprises the following steps:

(1) extracting total RNA of plants;

(2) denaturing the dsRNA into single-stranded RNA by heating the total RNA;

(3) carrying out efficient polyadenylic tail addition (namely A tail) on the 3' end of the single-stranded RNA to obtain total RNA added with the A tail, and purifying and recovering the total RNA added with the A tail;

(4) using oligo (dT)₁₀Reverse transcribing the total RNA added with the A tail into cDNA by RT, and purifying and recovering the cDNA;

(5) and uniformly dividing the genome or the genome end of the single-segment or multi-segment dsRNA virus into a5 'end region and a 3' end region to perform Multiplex PCR, recovering a target band after running gel, connecting the target band to a cloning vector, sequencing, and finally obtaining the genome end sequence of the dsRNA virus.

Further, in the above method, the heating conditions in step (2) are: quickly inserting into ice at 90 deg.C for 3-5 min, and standing for more than 10 min.

Further, in the above method, the oligo (dT) in the step (4)₁₀Primer sequences for RT are as follows: oligo (dT)₁₀-RT：5'-CACGCTCTCTACAGTCCGACGATCTTTTTTTTTTVN-3'; the 5 'and 3' ends of the dsRNA virus genome are treated by oligo (dT)₁₀The 5' -end of the cDNA after reverse transcription of the RT primer contains a common sequence 5-CACGCTC TCTACAGTCCGACGATCTTTTTTTTTT-3'。

Further, in the above method, the 5 'end region and the 3' end region in step (5) are each subjected to Multiplex PCR in the following reaction system: purified cDNA 9. mu.L, 2 XMultiplex Buffer 12.5. mu.L, 5'-mix primer or 3' -mix primer 2. mu.L, Anchor₂₄1. mu.L of F primer, 0.5. mu.L of Multiplex DNA Polymerase, 25. mu.L in total; wherein, the Anchor₂₄-the F primer sequence is: 5'-CACGCTCTCTACAGTCCGACGATC-3', respectively; the 5'-mix primer or the 3' -mix primer is as follows: designing a primer according to a sequence with the length of more than 100bp in each single segment or multi-segment of the dsRNA virus genome; the 5'-mix primer is formed by equal-volume mixing of 5' -end primers of each segment with the final concentration of 0.2-0.4 mu M; the 3'-mix primer is formed by mixing the 3' -end primers of each segment with the final concentration of 0.2-0.4 mu M in equal volume.

The method is applied to sequencing the terminal sequence of the plant dsRNA virus genome.

The invention has the advantages that:

(1) compared with the traditional method, the method has low requirement on the total RNA quality of the sample in the whole experimental process, does not need to specially extract dsRNA, only needs to extract the total RNA by using the conventional RNA extraction method, and can be used as an experimental material for identifying the terminal sequence of the dsRNA genome.

(2) The method of the invention heats the total RNA to denature the dsRNA into single-stranded RNA, then adds A tail to the 3' end of the single-stranded RNA, and finally adds A tail to the end of the dsRNA virus genome.

(3) Compared with the conventional method (identifying a terminal sequence requires designing a corresponding reverse transcription primer, and only the corresponding terminal sequence can be identified from the cDNA after each reverse transcription), the method of the present invention only needs to use oligo (dT)₁₀the-RT primer can completely invert the terminal sequence of the plant dsRNA virus into cDNA, the method can maximize the utilization rate of an RNA template, simplify the reverse transcription times, effectively reduce the cost of the primer and the use cost of reverse transcriptase, and effectively improve the work efficiency of experiments.

(4) According to the method, the ends of the dsRNA virus genome are uniformly divided into a5 'end region and a 3' end region to carry out Multiplex PCR, the 5 'end or the 3' end sequence of the virus genome can be amplified at one time through one PCR reaction, and a cDNA template can be maximally utilized in the Multiplex PCR. The method can quickly and conveniently obtain the terminal sequence of the dsRNA genome, and is very suitable for obtaining the terminal sequence of the dsRNA virus genome in large batch and obtaining the subsequent whole dsRNA genome sequence.

Drawings

FIG. 1 is a flow chart of a method for obtaining a plant dsRNA virus genome terminal sequence.

Figure 2 is a schematic of dsRNA plus a tail.

FIG. 3 is a schematic representation of reverse transcription of dsRNA into cDNA.

FIG. 4 is a schematic diagram of amplification of terminal sequences of dsRNA virus genomes.

FIG. 5 is an electrophoretogram of genomic 5 'and 3' end sequences of the amplified three-segment dsRNA virus CCV 1.

FIG. 6 is an electrophoretogram amplifying the 5 'and 3' end sequences of the genome of a ten-segment dsRNA virus SRBSDV.

Detailed Description

The technical solution of the present invention is described in detail below by way of examples with reference to the accompanying drawings.

The invention provides a method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome. Firstly, extracting total RNA of a plant, firstly, denaturing dsRNA into single-stranded RNA by a method of heating the total RNA at high temperature, and then, carrying out high-efficiency polyadenosine tail addition (namely A tail) on the 3' end of the single-stranded RNA to obtain total RNA with the A tail; after purifying and recovering it, oligo (dT) was used₁₀-RT reverse transcribing a-tailed total RNA into cDNA; after the cDNA is purified and recovered, the genome end of the single-segment or multi-segment dsRNA virus is divided into 2 parts (5 'end region Multiplex PCR and 3' end region Multiplex PCR) by a Multiplex PCR method to carry out PCR, a target band is recovered after glue running, the target band is connected to a cloning vector to carry out sequencing, and finally, the genome end sequence of the dsRNA virus is obtained (the flow chart of the method is shown in figure 1). The invention has low requirement on the total RNA quality of samples, and can simultaneously, quickly and massively obtain the dsRNA virus genome sequences or the terminal sequences of the dsRNA virus genomes in different regions.

Example 1A method for efficiently tailing and rapidly amplifying the genome terminal sequence of the tea tree CCV1 virus

1. Extracting total RNA of tea trees: tea tree total RNA is extracted by a CTAB-LiCl method (the temperature of the whole process of centrifugation is 4 ℃).

Total RNA plus a tail:

taking 10 mu g of total RNA, incubating at 90 ℃ for 3-5 min, quickly inserting into ice, and cooling for more than 10 min to obtain denatured single-stranded RNA. It was then subjected to a polyadenylic tail reaction (schematic of the A-tailed scheme is shown in FIG. 2). The reaction system is as follows: 10 XPoPoly (A) Polymerase Buffer 2. mu.L, ATP (10 mM) 1. mu.L, total RNA 10. mu.g, Poly (A) Polymerase (5U/. mu.L) (available from Vazyme, cat # DD 4111) 1-2. mu.L, supplemented to 20. mu.L with RNase-Free Water (pH 8.0). Reaction conditions are as follows: after the reaction system is fully and uniformly mixed, incubation is carried out for 50-60min at 37 ℃.

Purification of total RNA plus a tail:

the reaction product of the previous step was purified using RNA purification magnetic beads (purchased from Vazyme, cat # N412-01), and 2.2X magnetic beads were used, and the detailed procedure was described with reference to the reagent manual. Finally, 16 ul RNase-free ddH is used₂O (purchased from Vazyme, cat # P071-01) eluted the purified total RNA with A tail, and the purified product was subjected to the next experiment or stored at-80 ℃ for further use.

Reverse transcription of purified total RNA into cDNA:

the reverse transcription system is as follows: oligo (dT)₁₀1 μ L of-RT (5 '-CACGCTCTCTACAGTCCGACGATCTTTTTTTTTTVN-3'), purifying 15 μ L of total RNA added with A tail, mixing, heating at 70 ℃ for 5 min, rapidly placing on ice for quenching, and standing on ice for 2 min; then 2. mu.L of 10 XTT Mix, 2. mu.L of HiScript II Enzyme Mix (available from Vazyme, cat # R212) were added and mixed. The reaction time program of the whole system is 50 ℃ and 60 min; 2 min at 85 ℃; storing at 4 ℃. A schematic representation of the reverse transcription of purified total RNA to cDNA is shown in FIG. 3.

And purifying the cDNA:

the reaction product of the previous step was purified using DNA purification magnetic beads (available from Vazyme, cat # N411-01), and 2.2X magnetic beads were used, and the detailed procedure was according to the reagent instructions. Finally, 30 ul RNase-free ddH is used₂And O, eluting the purified cDNA, and carrying out the next experiment on the purified product or storing the purified product at the temperature of-20 ℃ for later use.

、Multiplex PCR

Multiplex PCR was performed on the 5 'and 3' end regions of the genome of the tea plant CCV1 virus, and the amplification scheme is shown in FIG. 4. Primers for Multiplex PCR of the 5 'and 3' end regions of the genome of the tea plant CCV1 virus are shown in Table 1.

TABLE 1

The primers involved in the invention are synthesized by the Beijing Optimalaceae Biotechnology Co.

The method comprises the following specific steps:

1) 5' end region Multiplex PCR amplification:

primers of CCV1-5'RNA1, CCV1-5' RNA2 and CCV1-5'RNA3 at final concentrations of 0.2. mu.M each were mixed in a volume ratio of 1:1:1 to 5' -mix primer. Multiplex PCR System: purified cDNA 9. mu.L, 2 XMultiplex Buffer 12.5. mu.L, 5' -mix primer 2. mu.L, Anchor₂₄F1. mu.L, Multiplex DNA Polymerase 0.5. mu.L, 25. mu.L in total.

2) 3' end region Multiplex PCR amplification:

CCV1-3'RNA1, CCV1-3' RNA2 and CCV1-3'RNA3 primers were mixed in a final concentration of 0.2. mu.M each in a volume ratio of 1:1:1 to form 3' -mix primers. Multiplex PCR System: purified cDNA 9. mu.L, 2 XMultiplex Buffer 12.5. mu.L, 3' -mix primer 2. mu.L, Anchor₂₄F1. mu.L, Multiplex DNA Polymerase 0.5. mu.L, 25. mu.L in total.

3) Reaction program of PCR instrument: 5 min at 95 deg.C, [ 30 s at 95 deg.C, 90 s at 60 deg.C, 60 s at 72 deg.C ] this step is cycled 35 times, 10 min at 72 deg.C, and stored at 4 deg.C.

The PCR products were run on agarose gel electrophoresis at a concentration of 1.5wt% to 2.0wt%, and the results are shown in FIG. 5.

Ligation, transformation and sequencing:

1) connection of

The band of interest was ligated to the cloning vector for Multiplex PCR run recovery. Reaction system: PCR product 4. mu.L, pEASY-T5 Zero Cloning Vector (from Beijing Quanjin Biotechnology Co., Ltd., cat # CT 501) 1. mu.L. The reaction was carried out in a PCR apparatus at 25 ℃ for 15 min.

2) Transformation of Escherichia coli

Trans1-T1 competent cells (50 μ L per tube) were removed from-80 ℃ and thawed on ice; adding 10 mu L of the ligation product when the competent cells just melt, and lightly shaking and uniformly mixing; gently centrifuging to make the liquid fall to the bottom of the tube, and carrying out ice bath for 30 min; thermally shocking at 42 deg.C for 90 s, and standing on ice for 5 min; adding 600 mu L of antibiotic-free LB liquid culture medium into an EP tube, putting the tube into a 37 ℃ shaking table, and performing shake culture at 200 rpm for 15 min; sucking 200 mu L of bacterial liquid by a liquid suction machine in an ultra-clean bench, uniformly coating the bacterial liquid on an LB solid flat plate containing Amp (100 mu g/mL) resistance, drying liquid on the surface of the flat plate, sealing the flat plate by using a sealing film, and putting the flat plate into an incubator at 37 ℃ for inverted culture for 10-12 h.

3) Sequencing

Single colonies were picked to 200. mu.L of Amp (100. mu.g/mL) resistant liquid LB, and cultured for 4 h at 37 ℃ with shaking table 200 and 220 rpm. And (3) putting 1 mu L of bacterial liquid in a10 mu LPCR system, and using universal primers M13-47 and M13-48 as primers to carry out positive clone identification.

Sequencing: sequencing was carried out using universal primers M13-47 and M13-48, which are well-known to the sequencing company, and finally the terminal sequence of the genome of the tea plant CCV1 virus (dsRNA virus) was obtained.

Specific nucleotide sequences of the obtained CCV1-5' RNA1 terminal sequence, CCV1-5' RNA2 terminal sequence and CCV1-5' RNA3 terminal sequence are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the specific nucleotide sequences of the obtained CCV1-3' RNA1 terminal sequence, CCV1-3' RNA2 terminal sequence and CCV1-3' RNA3 terminal sequence are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Example 2 method for efficient tailed rapid amplification of southern rice black-streaked dwarf virus (SRBSDV) genome terminal sequence

1. Total RNA from rice was extracted using Trizol (available from ThermoFisher, cat # 15596018), and the detailed procedures were performed according to the instructions of the reagents.

2. The primers for amplifying the terminal sequences of the southern rice black-streaked dwarf virus (SRBSDV) genome are shown in Table 2:

TABLE 2

1) 5' end region Multiplex PCR amplification: 10 primers, each at a final concentration of 0.4. mu.M, SRBSDV-5' RNA1 to SRBSDV-5' RNA10 were mixed in equal volumes to 5' -mix primers.

2) 3' end region Multiplex PCR amplification: 10 primers were mixed in equal volumes to 3' -mix primers for SRBSDV-3' RNA1 to SRBSDV-3' RNA10 at final concentrations of 0.4. mu.M each.

The rest of the procedure was the same as in example 1.

Agarose gel electrophoresis of the amplified southern rice black-streaked dwarf virus (SRBSDV) genome end sequences is shown in FIG. 6.

Obtaining specific nucleotide sequences of SRBSDV-5'RNA1 terminal sequence, SRBSDV-5' RNA2 terminal sequence, SRBSDV-5'RNA3 terminal sequence, SRBSDV-5' RNA4 terminal sequence, SRBSDV-5'RNA5 terminal sequence, SRBSDV-5' RNA6 terminal sequence, SRBSDV-5'RNA7 terminal sequence, SRBSDV-5' RNA8 terminal sequence, SRBSDV-5'RNA9 terminal sequence and SRBSDV-5' RNA10 terminal sequence, which are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16; obtaining the terminal sequence of SRBSDV-3'RNA1, the terminal sequence of SRBSDV-3' RNA2, the terminal sequence of SRBSDV-3'RNA3, the terminal sequence of SRBSDV-3' RNA4, the terminal sequence of SRBSDV-3'RNA5, the terminal sequence of SRBSDV-3' RNA6, the terminal sequence of SRBSDV-3'RNA7, the terminal sequence of SRBSDV-3' RNA8, the terminal sequence of SRBSDV-3'RNA9 and the terminal sequence of SRBSDV-3' RNA10, which are respectively shown as SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO. 26.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> method for efficiently tailing and rapidly amplifying terminal sequence of plant dsRNA virus genome and application thereof

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<211> 372

<212> RNA

<213> terminal sequence of SEQ ID NO.4 CCV1-3' RNA1

<400> 32

gaguaucaga augcucgcaa ugaacuggcc uguuuaagga uguuaaugua uccggaauac 60

gaagugguau cuggugauau uucugcuuug agagcaaaga guaucugccg cgacgccgga 120

aucaacuccc guuaucuguu ugaaauauuu auuuaucuca aagagaaaua cgguuuagcu 180

gagucucuuc ccuuucauuu gaaagucugg gacccaaccg aauuugaagc acgaagaauu 240

ucugcuucug aaauuaugua acgcaauuua uuugcaauaa aacuacgauu uuacaaucau 300

uuauuuuaaa uuuauugugu cgcauuauuu ccuuacacag ugguguagug guuuaaugcg 360

acacaacaug uc 372

<210> 33

<211> 813

<212> RNA

<213> terminal sequence of SEQ ID NO.5 CCV1-3' RNA2

<400> 33

caacacuccg ccaacuuauu ccaaggaucu ugaauuacca cucccuuuug cugacgcaau 60

acagaauuuc gguauauuug aaacccguug caugaagaag aauuuucuua ugauuccugu 120

guauccugaa gguaccaaga augaaggccg cgagaaagaa gacuggcacg cguaugaaua 180

cgaagccugc augcccaucc uccgcgagaa cggcauuccu aucaagaaug uugacacccg 240

ugugaaaucu ggaucugcau gguggacuua cagacucaag agugucaaug acacuuauga 300

uuuugaaugu auuuugccau uccugcauua cucagagcac ucagcucuac uuucaagcau 360

guuuuuacgu guucagagug gagagauacg cccaauuauc gagcucaaag gcgacgaugu 420

ugcauacggc guucgacugc gcgaaguaaa gcaaggauuc caacuccgag uguuuucagc 480

ccucugucac gcucacagcg aggaguggaa uuccuccgcu aauauugauu agccuugaua 540

acaauguguu uguuucccaa gaacaauuau guuuuguuau uucguauuag acccuacuug 600

ugaaagcuuu ucauuuugua uuaauuuuaa gucauuuuca uccauuucau guacuuuucu 660

uucaaucaau gaaauauuuc cuacuuucau augucuaucc aauugauuuc aucaaucaau 720

caguacaaca ccaaguguag gucccucgca ucaagauguc ccuugcggga ucgguucgcc 780

gaguuuugau cgugggcaca cuacaccacu auc 813

<210> 34

<211> 751

<212> RNA

<213> terminal sequence of SEQ ID NO.6 CCV1-3' RNA3

<400> 34

ccauacgcgc uugccauuuc ucagcuagga aucguaagag cuaguaguuu gauugaagag 60

aaaaucuucu gucccacaau ccaucaagga ucugcugcca auuuuuguuu accccaaaau 120

auucauuggu ccacugcuag guauuuacaa gcuguugagu uuggucaaaa gauaggaaug 180

cgguuugcac ccguugaccu aucggugcca uugggcucca guuggugguu auaucgccag 240

gaugauacag auagucuguu uucccugcaa uguuccaucc cugaggacaa uuucacugaa 300

gcaacugccg uccugcacag uuuauuuugc auuaacgagc aaggugguuu ugcaaacgag 360

aucuuuaacc ugaauccgau ugaaaggcau gauuacggug cuaugcuccg caacccucac 420

gccgacauca augucaguag cuacuucgcg aucgaagacg cuccggaaac aguuuggaaa 480

gucguguaau uugaaauguu uuuacauuuu guuacgaacg auaauggacu guuauuuuua 540

aguuguugua gaacucuuau uuugauaacu guuuuaauuc ugaauacuuu uacuuuugaa 600

uuaauuuggu gugugauuau auauuuaaau caacucuaua cuugacaguu guacuaacag 660

agcgacuacc ggaaggguag agcucaauca aguguaggug caacuaagau ucguuuacga 720

aaaugauuag uugccacacu acaccauucu c 751

<210> 35

<211> 1183

<212> RNA

<213> terminal sequence of SEQ ID NO.7 SRBSDV-5' RNA1

<400> 35

aaguuuuuuu cgccgaccua cguaucucau agacaaaugg uggaacgaaa guucaguaga 60

ucugcagaaa uuugcuuaaa aaaauucaac ucaauccaaa aauuuagaca aaacaagcaa 120

cagcaaaaaa ccaacagaga ccgaacaauu ucucaaaaag aaaagaaaau aaaggacaga 180

gccccucccc cuucaaagaa agaagacauu cccacaauuu cuaccccuau uucaauugaa 240

ucucaaucaa auuuggauga acaaagaaca ucuuacauuc aucuuuuaca ucaaaaagau 300

uccauuuuag acgcaauuau ugaagaugcu gaaaacauuc gaaacguuuu uauuggugaa 360

gugccuuuaa ccaaagaaau cauuaaaaau cgacuagaug acauuucgac aguguuaaau 420

aaugcgagug auuugauucu caaauuauuu gaaucugaua augguauuuu gaaugaaugu 480

guuaucccga augacguuau acaaaaagau auuuauucca uuuugcaucc cucugaaauu 540

uuugauuaua augguuuugg cauugaugaa guuaauaacu uagcgauuag uccaaagauu 600

agauauuuac cuaauuggaa cauucagaaa auuagaagua cuauugaucu uuugaaaaac 660

gauaauucuc cuuugaaacc agucaaauug gcucaugauu augguuugau uaguaauaac 720

uuaaaacgua aauuauuaau ugguguugau agaauguuua auaaauuaag uauucaacau 780

agauuuggua cuuuugaaag ucaaauauuc aguuguuuuc uuaugaaaca auugcuaaca 840

gaagaacagu guuacgucaa ucuuccaaau ugcguuuuaa aaucaaaauu ucaaaaaaga 900

ggaauugaca cuguaugcug gcaauauuug cgacaaauca uccuucauua caaugguuuu 960

ccuuuuuauc aucacacuca uagugcaaga uuuggugugc uugacaguac aaaaauuucu 1020

auucauaccg auuuaccagu uucuugugua cuuccaagug uuuuacaugg auuguuaaac 1080

ggcauaguuu cagaauuauu augugauuug aacucagaga cagcuuuaau uuaugccaaa 1140

cauuugaugu uuugcucuca acaaucaacu uaucaacguc aac 1183

<210> 36

<211> 211

<212> RNA

<213> terminal sequence of SEQ ID NO.8 SRBSDV-5' RNA2

<400> 36

aaguuuuuua ccggacccuu ggauucaccc aaaauuaagg acgaaaugaa uuuggaggag 60

aaccguaaaa aaggcgcuaa acaagaugaa gaugaaaaga aagaaccaga uaaaacgcaa 120

gaagcagaag aaccagcuca agaaaaacaa gaaucacaga aaagucaaga gaaaacugac 180

ucagucgauc cugacgcugu cauuaaucaa g 211

<210> 37

<211> 1550

<212> RNA

<213> terminal sequence of SEQ ID NO.9 SRBSDV-5' RNA3

<400> 37

aaguuuuuau ccaugucuuu gaauugaaca gggauggauc caguacaagu caucaaauac 60

cuuuugucuc aaaauuauga aaccaaauua caacaacucg aacaauggca acaacaaaug 120

caagcagcag gcuucacaac agaauacuua caacgaucuu cgaacgugcu aaacauuaga 180

cgcagaauug aagaaucuag cgggcguuua uucgaaauca gugcuaaacu auggcaaggu 240

agucuauuua cuaaaaccau uucuccuaau aauuuauuuc ucggcuucag ugaaugcuca 300

ggagugaaca aaucgcuuuu ugauucuaaa aauugugauu uuggaagauu caguuuaaac 360

uuucauugca uaccuaaaua uuggauauua ucuggaauau uuaaugauuu aguuuguuau 420

gaucaucaua augucaaagu cacaguaagc aaaucgucaa auaaaauauc auuuagaaua 480

aaugacuuua uuuacauagu uccaaccaua agcaacuucu cauuuuuauc ugacaaucca 540

gauaucaaaa uugaugaaaa cugcgcauuu gacguauuug uacgaauuuu ucaaucauua 600

agcgauguug uuauagacgu uuguaauuuu acauuugacg augaauuauu gaauguugaa 660

uggaaaaaca caaucauucg uuuugccggc gaugcuaacu auuucucagu uucuucaaac 720

cucauuuuca auaguauuga cgauuauuua gguagaaaau gcacuucaau gagagauuau 780

uuuuguuacg caauuaguau gaauaaccug gaaaugauua augacuccuu aaauaauugg 840

gaauccaaac aacugucaca uaaaaagaau uuaguaaaca gucuucauca aaugaaucuu 900

aauucaauga uuaaaauuuc uucuuuggaa aaggaaguug cuagaguuaa gacguuaaaa 960

auauccuuaa guuauccucg uacgauuugu uugcacauuc caacgcauaa gcuuuuuauu 1020

aauaaguauu cucaugacuu auuuucuuca guuuuuaaga cucaauuugu ugguuggaau 1080

uuuacauaug gugaaagaga uagaacuguu gaaccuuuca uuugugaugu ugaaacaaaa 1140

cuuaaaacga auucuuccau aguaaguaca gcaucaacuu cucacuuuca auguaaugau 1200

uuuggcaguc cugauaacau guuuuugcua aauguuuuag auaaggaugg uaacguuaaa 1260

aaaguaauuu gcgaagcugu guuuucauua aaguacaaag aguuuaaaua ucugggaguu 1320

aaaucucucg augaaaaaac aggauaucaa agucaaguuu auuucuuugg uuugagugaa 1380

auuaggcuuu auacugaaaa uccaaaguug cagguucuuu cauuagugag ugaacaugaa 1440

gguaaauuau gucauguauu uuaugcaacu acuucuuuaa uuuuaucuuu caaaauucug 1500

gcugaaaaua ucgaauauuu aggaguuacg ucuccucaau ucacaagcga 1550

<210> 38

<211> 502

<212> RNA

<213> terminal sequence of SEQ ID NO.10 SRBSDV-5' RNA4

<400> 38

aaguuuuuuu caggauguug aaaguaaaug ugcaggcccu cauuuauccu uucagcggac 60

aaauuacaga cauuagaaga aaccaacuca uuccaacugc uaaauucaaa cacacugucu 120

uacaucagcc uauuuuuauu acaaaacuua aaaauaaauu cucauccaau gacgccccuc 180

caacucgaaa cacucuacga guuaauccug uuuggucaaa ugcuuuuuuu gauuggaauu 240

cagcuuuauu aaauccuugg acuguccuuc ccuccucgac uggcucuggc ucaaugucaa 300

uuccaucaca uaaaaauguu gaaucaggca acgcaauuuu gaaaaaugcu gaauucauua 360

acgaacgucu uccagcuuac uuaaagcugu uguuugauag uuugaauuug aaagaaaaug 420

gauauguuaa ucaauuuggg uuuugggcuu uauuauaccu agauuacagu uugcauuguc 480

ucguaccaac ugaauauuau cg 502

<210> 39

<211> 374

<212> RNA

<213> terminal sequence of SEQ ID NO.11 SRBSDV-5' RNA5

<400> 39

aaguuuuuuu cacucaugac auauuugaaa gugaagauga ccacagaucc uuucgaaaug 60

uuuuugaaaa aagugguuuu caacccaacg aacgaaauuc gaauucucaa ccacgaacaa 120

gcagacaaca cucuuuugac cacacugaug auuauucacg acuuugaaga cacccccacu 180

cgugacucaa uuuugcccca aauugucaau gaagaaaaag ccgaauucga ccgaacuauu 240

gaauccauuc uugaagaagu uagcgaugcc uuuguugcua auuaucacaa aauucucgaa 300

gaaaacgcga aauuggguaa cccucuugcu ccaaauuaca aaaacgucgu gucuguagaa 360

acauguauuc cagg 374

<210> 40

<211> 342

<212> RNA

<213> terminal sequence of SEQ ID NO.12 SRBSDV-5' RNA6

<400> 40

aaguuuuuug agucugagau acuguuaucc auugcgaacc ugagaacgag uuuaacagaa 60

ucucacaccc agaaaacaac gaugucuacc aaccucacga acauagccau gcugaaaaaa 120

cgauugcaaa gacgagucac cacuaacacc ucugucaaca augaagucga gaacacucca 180

ucccaaccac ucgaaccugu ugcgaacgcu ucuucuucuu cagaaagcuc cgucauuacc 240

aaacguuacg cccguccguc uaacaaaauu ucuucuucau cuacaguugc cgagaauuca 300

aaugauacga aacugcucca caacuugauu uccgcagcgc ca 342

<210> 41

<211> 647

<212> RNA

<213> terminal sequence of SEQ ID NO.13 SRBSDV-5' RNA7

<400> 41

aaguuuuuuu cgaccugucu ggaccagaac auaaaagaac auggauagac cugcucgaga 60

acauuuaaaa uucaguaaag cgaacacuaa aaacgaaaua caagaaauga gaauuuacaa 120

agacgacacu gcaaauggau uauguuucag cgaaauuaac guaggaugca cuuccacuuc 180

acccaaaaug gcucuuuccg acuacuucag uuccguuagu uguaguuuug auggcgaaau 240

gcguacuccc gaccuuccuu uacauuuaua uggugaucuu cauuuucacg accaauuuac 300

uaaugacgua gauuuagauc uacuuuguug gcaauuauug aguuccaauc aagauucaag 360

agcuuugugu guuaauauuu uaagaaugcu aacugcacuu ucuuuaggaa acgcuuuuau 420

uucggaagga cgauaccauu augcacuuga cacuacugaa acuacuucau cugaagaugu 480

cgacgcacua cgauuacuau cacguuuagc uaaaauaguc auuaaaaaua auuugaaaag 540

cgaagauguu acuuuagcuc aacaaaauuu aauuuauuau uauuuuggca cuagcuuuaa 600

aggcauucau uuaaauuggg auucuagauc uagucaacca agcguac 647

<210> 42

<211> 888

<212> RNA

<213> terminal sequence of SEQ ID NO.14 SRBSDV-5' RNA8

<400> 42

aaguuuuuuu cgcacugucu aaagaugauu ggcaccuaug augaccggau uucuuuaaga 60

cuauuauucu ucaacuuaug gauuaaaaaa cgacccaaug acccaaucaa ccaucgaaaa 120

acgaagaaaa aacagaaaac gauacaugaa aucgcaacuu ugacugacca uuuagucucu 180

ccuucccuuu cugaauaugc ggaacaacua gcagaugagu uugcgaaaac uaaacauccu 240

aucuuuacac cuucucaauu auuuuccacu uucacgcguc uagaaacgau cuuuaaccga 300

gcaggccauu uuccugauuu auuuagcacg ccuauuaaau accuuggauu cuugauuuuc 360

augccugacu ccucaguucu cuucgaccca cccacuccag aacauucuua uuacaauuug 420

aaaaagaauc auaaaaugag cuucagugaa uuggaaacau uguuaacugc gaaagcauug 480

acaggucaaa uuauuaaaac ugaaauaaau gauuuugcug auaguuuuga aacuuauuau 540

uacgauccca gaagugagag agcuaguuuc ggcuuuuuga gaagaacagc cauggugaga 600

uuugauuuuu uauccuacaa auauaacaau gauuugaccg uaacugaacg ugaaaaauua 660

ccaacuuggg cuuccaauuu gaucacucgu augaauguug uaaacacuuc ucuuccuuac 720

gaaauuaacu guggaaauau uaauguaaau cagcauuuug cuguaauagu uuugaaaaau 780

gcuaagacga auguuacucg agcuagauuu uggaccguau uggacgauga gcgcaacuuu 840

guuuccauaa aaauggaucu ucaagcuauc accuuugaac guaacgau 888

<210> 43

<211> 777

<212> RNA

<213> SEQ ID NO. 15: SRBSDV-5' RNA9 terminal sequence

<400> 43

aaguuuuuua agccuggaac ugacacauuc cugcaggaac gaacgcaaga gaauggcaga 60

ccuagagcgu agaacguuug gaucauauaa aauagaagaa uugacaauua gaaacgacca 120

accaacaaga aauacaaacc uuucacuuuc ucaaucgacc gagaaucguc uuucaaccaa 180

gaaaauuccc cuacucgacg acggcauuuu ugaacuuuug aacuacuuca uugauggaac 240

caauuucaac aaaacguguu auuguggauu uaauuauucu caucucccaa aucuagaacg 300

ugacuuuaac aucgcuucuu uguauguacg ugaaaauuuc gaaauuugca cagaucaauu 360

ggacuuggcu aacuacguuc gacaaccgaa caucagcauu aaaucuccug acuucacagu 420

uuguuuggaa uacguuuuga aaauugucgu ugaaagcgaa uccucaacua aagaccaaaa 480

agaugaugaa ucacagagac ccaccuccac ugauucaacu aagaaugagc aagaaaagaa 540

guuuguugaa augucuuugc uuccucuguu aaaucgcgaa ucugaagaau cuuugacuga 600

agaaauuuua gaaggugaag gcgcaguggu aaacguacuu aaacuauuca uuaaaggauu 660

uuugaugcau uuaggugaga auccaaauuc uuaugaucgu caacuaaccg uugaaaaaua 720

ucguccauua uugguuucua uuguuggcua ugaauaucua gucgguacua caguucc 777

<210> 44

<211> 295

<212> RNA

<213> SEQ ID NO. 16: SRBSDV-5' RNA10 terminal sequence

<400> 44

aaguuuuuuu ccucauccau aauggcugac auaagacuug acauagcgcc cgaucuuauc 60

cauaauggug ugccucaaag acuuucagau acgauaauuc uaaacaaccg accaacaauc 120

acacuucugu cucacuucaa cucucuuuuu cacgaaucaa auauuguuaa aucaccccac 180

aucgcgucau cucaaacuac aguuaauuua uacauucgua agcauuuacu gacucgacuu 240

caugauagau uacaaacugu cgaaaccagu acucuaccga acauuacuca acuua 295

<210> 45

<211> 963

<212> RNA

<213> SEQ ID NO. 17: SRBSDV-3' RNA1 terminal sequence

<400> 45

ucagugcuca aggcucacaa gauugaagug ccuuacggau uaauguacgc gaaucauaau 60

agaucaaaaa ucgaucaauc uuugacuguu agaacagaau cucucgaaga aaggguauau 120

uuggauucua uauuuuugaa gcauaugcua gauuauaaaa cugugccuaa agauuuagaa 180

guacugaaga cucaugcucu cuguuccguu cguuuaagaa uuuaugguga uuuugauuau 240

gcuugugaaa aagauggugu uaucgaaccu aguucugacg cucaguucaa uaucauuuua 300

ccuuuuuuac cugguuauca uuuaaacagu gcuuauggca aauuauuuuu guauuccacu 360

uugcccaccg ugcaugauag agauauuucc gguacuuuag gagaaauaac aggaucuuua 420

ggcgcuucuu uugauguaga ugcugcgaua gaauuuggug cacauguguu uaauauuaau 480

ccaaguuugg uugaugccgc aggcacugcu auugggauuc cucaaggauu auugaaucau 540

uauaaaaauu ugguuaacgc uuuuguuacu aauaacuuca auuuaaaaua ucauuccaua 600

uuuuuaaacg uaaaauauuu ugguuuaaau ggcaguuuaa aacauuuuuc uacuuuugga 660

gauuauucaa gucgucuugu ucguuuugac uuaauacaua aguuggauag auaucauaau 720

guuuuuguac gugauuucau uuuugcuuau auucacgaac uuaaugggcg cagaguguau 780

cucgauuaua guuugcauag ucugcuguua gucaugucuc gaggaucuau aaaguauuuu 840

acaucucauc ccucucaauu auuuaauccu augcuuacuc uugaguucga cugauaaggc 900

guucccgauu uugaaacuau gaagugagcg ugguuagugu ccauggcgaa aacagcuguc 960

guc 963

<210> 46

<211> 846

<212> RNA

<213> SEQ ID NO. 18: SRBSDV-3' RNA2 terminal sequence

<400> 46

ugguuauuac aguaccggca caucuucacc cgcagacuuc uuuaacgucg aaaacgauac 60

ugaauauuuc acuauuaauu uagacgauca uccugaagug uuuaccacca cugguacaga 120

cgguuucagu guucaauuua aauucaaaaa agacaacguu gaucacaaau acauuaaucc 180

acuaccuaac gcuauuccua aaauaaaauu uaucuugaag agauuaggua auaugaauga 240

acauuuugua auuuucauga aaagaaugau ugauacuaga agacauaugg uaguucuaac 300

cgaugugauu guugacuaua acauugucac uuuagccaau uggaaagacu uuaagauaag 360

ugacgagaac aucuuuaacc aauuuguuuu gaaucgugau cugaauaaca uucaacuaaa 420

cuuuuacgau acuagauuca uuauucaaaa ucguacugau gaauugcucu accguuacau 480

uagcuuaagu uaucguaaca uugccguuuu aaaagacuuc guaguaaugg gagguauuuu 540

ugguaauccu aaccaggcca uugacaaauu gacugagaua gauaaagacg uauacuguuu 600

uggagaacgu aauggucaua cuucaaagua uuuuaucagu aaaaaagaca agauuaaagg 660

aaguaccguu aacuuuaaca aucauuucac uuaucuugga ccacaaucug agacuagacu 720

gcccauuuug gauaucagag uuucaucuca aauuuaauua acaucuuucu ccuucuuguu 780

cgaugugucu agcuuuuugc gguguauucu aauggucguc cacaaccggu aaaaccagcu 840

gauguc 846

<210> 47

<211> 213

<212> RNA

<213> SEQ ID NO. 19: SRBSDV-3' RNA3 terminal sequence

<400> 47

gcuauucauu uacgucaagu cgacucuacg auuuauaaaa ccaugcaugg acauucauuu 60

ucggaucaag uaccuaauga aaguuauuuu aaaaugauuu caacuauagu uucucaucca 120

gagauuuuga gauuuuaagg cacuuugguu agcacgcagc uggugucuuc cuuaugcagc 180

ugaaauucga cacacauggg uacagcugau guc 213

<210> 48

<211> 565

<212> RNA

<213> SEQ ID NO. 20: SRBSDV-3' RNA4 terminal sequence

<400> 48

cugaauugug cgguuuagga cacgucuucg cuuuuaauuc uuauuacaug gguuuuucua 60

cuagagacga auuggagaaa gaauugaauu acauugcuga uacacuagug auaaaagguu 120

ucuuuuucau aaguuuuuac gaaauggagg acgauuuaaa accuguauug aaacaucaug 180

gguuuauaga uauaacuagu gcggaugugg cugccuauaa auucacuuuu ggaaaauauc 240

augccgucgc uacugucagu guggauuuug uaaaaaagug gaaagcaaaa augaugacua 300

aauaugacgu uuaucaaauc uuuuuaucag cuaaugaugu uaguuucagu uguaucaugc 360

augguuauuc ugucaauuua aauaguaugu auuucgcgcc gguuuauaac uuauuuucuc 420

cuuguuucuu aauucauccc aaaaaaugaa uuuuuaaugu uugugcauga gucuugaaag 480

gauaugaaau acugccacau gcccucuucc cuacccuuuu aacauaccgg gugggggcga 540

auuaaaccug aaaacagcug auguc 565

<210> 49

<211> 1090

<212> RNA

<213> SEQ ID NO. 21: SRBSDV-3' RNA5 terminal sequence

<400> 49

cuggcucagu cacacaauac cucauaccug aauggcuugg agaaaguuug acgguuuacg 60

aagaaaaaca ugaaggcgcu cgucuuuuug aaaaaucuag cuuguuuuca acuuuaagcu 120

gcauuccggc aacguuuuua gcaaaucaca uacuaaucga auguuuagaa aaaagcucga 180

acaaacugau uucuuucaug gaugguguga ucagugccau guccacuagc aucgaauucu 240

cgacuuucaa aaauaccaug cucaacaacu ugaaaaggug uacacaggcu ucugagauga 300

acguugcgaa acagucucca aucgcaggaa cuagcaaaaa caagucuuuu cguuccuauu 360

uacgaucuuc ucgaccaaac caugucuaug acgcguuguc cuauuguauc uccaagaaac 420

agaacggguu guucacauca gaagaaaugu gcaugucuua uaaucucaga ucauuuggaa 480

aagugggagu ugacuauagc aacggugaau ugucauauga uaucucguca agagaagagc 540

uguacaacaa ucaucccugu gcgcuucgac uuagaaaucu cggacauuuu gucuuuggaa 600

aacauuuuag augcaaagua ugucgcaaca auuacggaac uagcuuugaa cuuucccugu 660

gucaauuggg acaucaagaa acagcaauug aagcaguucg auccaacaca aucacuaguc 720

aaauuagagc aaaacuucaa ccaacuacag cgcgaugaua agcuucauuu uaauuguugu 780

uauaauuauc cuacuuuaca cauauuaaac cguauuuuaa auguuucuaa agacguaauu 840

ccauuuuuau uugaucacaa ucacaacgcu aacaauaaaa cgaugucuga cuuaucuucu 900

acgcuuaaca auugucguga uaugaaaguu ucugagagac aucacuuacu uggaacuuua 960

cuuuucggaa gcacgcuagg aguuauacuu cauguuugag gguguguuau cccuggcugc 1020

aagcgggagg cgcgugaaca gguguuccac gucuucuuag augcgggggg gagugaauac 1080

agcugauguc 1090

<210> 50

<211> 483

<212> RNA

<213> SEQ ID NO. 22: SRBSDV-3' RNA6 terminal sequence

<400> 50

uggcguagau gcuaacuuug gcgaucugac uucacuauug cguaugcuuu ugaucaagau 60

ugauaguauc acuucacuuu gcgaugcgaa cguuacugug ucugagaacc aauggagcgc 120

guauggauua uguaguuuga cuucaucaac gcaugacaau gccacaacug aaaugcuguc 180

uguaaaucaa gucgguuuaa ugaauuucca aguugcgaac uucaccaaca ucguuuauga 240

cgucuuuuuu uacaacaugu ucauuaaguu uuuguggcaa cuuauuucag aguaaugaac 300

aaauaaucac gaaugacauc ucgcauuaac aucugcgcac uaccaccuag cugauuugag 360

uuauuauacc aucuuuaacg gacagagauc ucauguugaa cuggauuauc gguauucgag 420

augguaaguc aguuuucucu auucgucacc uugcucggca agagacucaa aucagcugau 480

guc 483

<210> 51

<211> 750

<212> RNA

<213> SEQ ID NO. 23: SRBSDV-3' RNA7 terminal sequence

<400> 51

gaaguuagug augaauggug cgacauugac aauuuuuuag auauuagagu uguaaaugau 60

gaaucucaau uugaauuugu uaauucccau auuaacaguc guuuauuaau uacgcuaaau 120

ucaaauccca acauuuugug gacugcaguu ggucuuuuaa cuaaaauuuc ucucuuacag 180

gaauuugaaa auuuugaaau uuuaaauuau uggcaagcga uggaaagaag augggauuua 240

augaaugacg auuuaaaaau aggauuuguu uuuagagcuu ucgauuugaa acaaaaucaa 300

uuugaguuac uuacuaaauu auuaggggau aguuuauuuu uugcuggcau uaauuuaauu 360

gguaagucaa guauguuacc aauguuaaca gugcauucaa uuucugauua cauagaucau 420

ugguuuccua cagaauguua uucaagugau aauuucaugu cuuuuauuaa augucacacg 480

auuaccguuc cuaaauggaa gaaaauuguc guucaauuuu auuuacggca aauauuuagu 540

agaagcagaa cgcaaguauu aauggcacau guggauauag aucauuggua ugauguuuuu 600

augaaaaccu ugguuuuuaa gaguaugaga aaaacgaaaa agauguugaa aaacauucug 660

aauuuguaau gucucgucga ggugguaugu ccuguuuuua auguuggugg aaucucagac 720

augugucaag gucgaaaugc agcugauguc 750

<210> 52

<211> 398

<212> RNA

<213> SEQ ID NO. 24: SRBSDV-3' RNA8 terminal sequence

<400> 52

guucauucuu acugugaauu agcguucgua ccucauucgc uggcauauau uacucuucac 60

aguaguuaua acuccgcagu uaucggaauu cuuccacgaa auggucaaag cguauuaagu 120

ccuacucgug ccauugcgaa ucagauguua cacagcuucu augaacgcua uacauccaac 180

augaauccca cucccgcuuu ucucuucuca uauuucuuug guuuaaccaa aggaaucaac 240

ccuuuaacag cugugucuau ucuuugcuaa auuauacuuu gaggaauaau acguccuuuu 300

uggguccgac ucauauaacg guguuucagu ccaauaucuu gauggcagac uaggucgaug 360

uagauucuag ugaagugugc gaaaauucag cugccguc 398

<210> 53

<211> 563

<212> RNA

<213> SEQ ID NO. 25: SRBSDV-3' RNA9 terminal sequence

<400> 53

uagauauuga agccaaaguc gauccugugg uaagaagaaa auacgguaaa guuggucaua 60

uaauguuaau gauuuuuagc uuucuuuucu uugggauuuu uaaauuaacg cuuaaaaugu 120

uuuaucaucu uuuucggugu guauguugua auccuuuaau ucgaggaauu uuuaguguug 180

uuuguacugu aguuuucuac auagucauuu uuacgauuau uuauuuaguu uauuuuuucu 240

uuggugauca aauccuugcu guauaucauu cuuugaccga gaugagcgau ucugguuuaa 300

ucaacuccac caaaaucgaa gaaaagguua acaacaucau ucacgaaggu ucuuuauuuu 360

uugguuccgu ugauccuaau acuggccacc uucaagaagu cgaaaaacaa guguucaaug 420

gagguacugu aaauuauacu uuguuucacu aaguuucuau uuuaugugac ucugaaaaua 480

uucacgcucg uaagucgauu uucuugcagu uguguauacg cuacccaaca aauucguucu 540

caagccggcu uacagcugau guc 563

<210> 54

<211> 256

<212> RNA

<213> SEQ ID NO. 26: SRBSDV-3' RNA10 terminal sequence

<400> 54

uguuaugaau ucaguuauua uuagcagagc caacaauuua uugaaggcgg aucgcgaucg 60

ucuaauuaag aaagcuacca cugcaaauuc aucuaccagu aauucaaauu cagaacaugg 120

ucaaaagauu guguuaaaua aagucaccag augauaauug agguuucauc auuagcgcga 180

cuaguucagu gcuguguggu cuucaguuca uuaucgacgc cgagugacga cggcuagggg 240

gaaagcagcu gauguc 256

Claims (5)

1. A method for efficiently tailing and rapidly amplifying a terminal sequence of a plant dsRNA virus genome is characterized by comprising the following steps:

(1) extracting total RNA of plants;

(2) denaturing the dsRNA into single-stranded RNA by heating the total RNA;

(3) carrying out efficient polyadenylic tail addition on the 3' end of the single-stranded RNA to obtain RNA with an A tail, and then purifying and recovering the RNA;

(4) using oligo (dT)₁₀Reverse transcription of RNA into cDNA by RT primer, purification and recovery of cDNA;

(5) and uniformly dividing the 5 'end and the 3' end of the genome of the single-segmented or multi-segmented dsRNA virus into a5 'end region and a 3' end region by taking cDNA as a template, carrying out Multiplex PCR, recovering a target band after running gel, connecting the target band to a cloning vector, and sequencing to finally obtain a complete dsRNA virus genome terminal sequence.

2. The method of claim 1, wherein: the heating conditions in the step (2) are as follows: quickly inserting into ice at 90 deg.C for 3-5 min for more than 10 min.

3.The method of claim 1, wherein: the oligo (dT) in step (4)₁₀Primer sequences for RT are as follows: oligo (dT)₁₀-RT：5'-CACGCTCTCTACAGTCCGACGATCTTTTTTTTTTVN-3'。

4. The method of claim 1, wherein: and (5) carrying out Multiplex PCR on the 5 'end region and the 3' end region respectively, wherein the reaction system is as follows: purified cDNA 9. mu.L, 2 XMultiplex Buffer 12.5. mu.L, 5'-mix primer or 3' -mix primer 2. mu.L, Anchor₂₄1. mu.L of F primer, 0.5. mu.L of Multiplex DNA Polymerase, 25. mu.L in total;

the Anchor₂₄-the F primer sequence is: 5'-CACGCTCTCTACAGTCCGACGATC-3', respectively;

the 5'-mix primer or the 3' -mix primer is as follows: designing a primer according to a sequence with the length of more than 100bp in each segment of single segment or multiple segments of the dsRNA virus genome; the 5'-mix primer is formed by equal-volume mixing of 5' -end primers of each segment with the final concentration of 0.2-0.4 mu M; the 3'-mix primer is formed by mixing the 3' -end primers of each segment with the final concentration of 0.2-0.4 mu M in equal volume.

5. Use of the method of claim 1 for sequencing the ends of the genome of a plant dsRNA virus.

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