CN110283883A - A kind of primer and method for unknown RNA mycoviruses genomic clone - Google Patents
A kind of primer and method for unknown RNA mycoviruses genomic clone Download PDFInfo
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- CN110283883A CN110283883A CN201910378238.0A CN201910378238A CN110283883A CN 110283883 A CN110283883 A CN 110283883A CN 201910378238 A CN201910378238 A CN 201910378238A CN 110283883 A CN110283883 A CN 110283883A
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Abstract
The invention belongs to gene engineering technology fields, and in particular to a kind of primer and method for unknown RNA mycoviruses genomic clone.The primer includes forward primer and complementary primer, and for nucleotide sequence respectively as shown in SEQ ID No.1 and SEQ ID No.2, this method, which carries out unknown RNA mycoviruses genomic clone using the primer, can get unknown virus dsRNA full length cDNA sequence.The forward primer designed in the present invention is that do not have similitude with fungi known and plant virus sequence according to Iresine herbetii viroid sequence design, avoids non-specific amplification when amplification virus sequence；The joint sequence is shorter than known joint primer sequence simultaneously, and joint efficiency is high；Reverse transcription is also added into random primer other than complementary primer is added in viral genome amplification procedure, it is ensured that viral genome reverse transcription is more complete, is adapted to the amplification of mycovirus rna genome, has broad application prospects.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind is used for unknown RNA mycoviruses genomic clone
Primer and method.
Background technique
Virus can be divided into plant virus, animal virus, bacteriophage and mycoviruses etc. according to the difference of host.Mycoviruses
It is widely present in disease fungus, asymptomatic latent infection is presented mostly and does not cause host's character mutation.Some mycoviruses
It brings about a wholesome effect to host fungi, the enhancing of host's pathogenicity or enhancing host fungi is such as caused to post the heat resistance with plant.
A small number of mycoviruses can cause host's character mutation, including cause the decline of host's pathogenicity etc..Weak poison Relative Fungi virus can be made
Plant fungal disease control is used for for potential bio-control factors.Meanwhile the interaction system of mycoviruses and host fungi is right
Solution virus and epiphyte pathogenic provide a good model and tool.In addition to this, there are fungies abundant in nature
Virus Resource Virus Resource, most mycoviruses have multifarious molecular characterization and biological property.So the hair of new mycoviruses
The understanding to entire virus evolution and ecology is now helped to improve, development virological to promotion has great importance.It obtains
Viral whole genome sequence is the genome structure of parsing virus and the key step of evolutionary relationship and research known viral gene function
Suddenly.
Most of virus has the genome of RNA, including dsRNA and ssRNA virus, and ssRNA virus is in a replication process
There is the replicative intermediate of one section of dsRNA form, so dsRNA and ssRNA virus can be obtained by extracting dsRNA in fungi
?.The method of conventional viral diagnosis and sequencing includes virion and nucleic acid being separated to out of culture fungi, by random
Primer synthesizes and establishes cDNA library, the segmentation gap obtained by design specific primer filling-in cDNA library, finally by
RACE method determines end sequence, or the end sequence of dsRNA is obtained with single primer amplification method, i.e., first in the 3'-OH of dsRNA
End plus the end of the preceding paragraph 5 ' are phosphorylated, and 3 ' ends are then reversed with the primer complementary with connector by amidized joint sequence
Record, and carry out PCR amplification with acquired dsRNA sequence design specific primer and the primer complementary with connector and obtain end sequence
Column.These method and steps are more, comparatively laborious, need by the intermediate design of primers stage, it is therefore necessary to develop a kind of effect
The method of the higher amplification viral RNA full length cDNA sequence of rate.
Summary of the invention
Against the above technical problems, the object of the present invention is to provide one kind to be used for unknown RNA mycoviruses genomic clone
Primer and method, mycoviruses dsRNA full length cDNA sequence can be quickly obtained, to solve mycoviruses detection conventional at present
More, the relatively complicated problem with sequencing approach step.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of primer for unknown RNA mycoviruses genomic clone, the primer include that forward primer and complementation are drawn
Object, the nucleotide sequence of the forward primer is as shown in SEQ ID No.1, the nucleotide sequence of the complementary primer such as SEQ ID
Shown in No.2.
Further, the forward primer and known viruse sequence are without homology, and 5 ' hold phosphorylations, 3 ' Amino End Groups.
Further, the unknown RNA mycoviruses include diplornavirus and single strand RNA virus.
A method of unknown RNA mycoviruses genomic clone is carried out using the primer, comprising the following steps:
(1) extraction of mycoviruses dsRNA；
(2) forward primer is attached using ligase with dsRNA, and connection product is purified；
(3) using the connection product as template, reverse transcription is carried out using the complementary primer and random primer and synthesizes cDNA
First chain；
(4) using first chain of cDNA as template, PCR amplification is carried out with the complementary primer, obtains pcr amplification product；
(5) cloning and sequencing is carried out to pcr amplification product.
Further, this method is for obtaining unknown virus dsRNA full length cDNA sequence.
Compared with prior art, the invention has the following beneficial effects:
The forward primer designed in the present invention is according to Iresine herbetii viroid sequence design, with fungi known and plant virus sequence
No similitude is arranged, non-specific amplification when amplification virus sequence is avoided；The joint sequence is than known joint primer sequence simultaneously
Arrange short, joint efficiency height；Reverse transcription is also added at random other than complementary primer is added in viral genome amplification procedure
Primer, it is ensured that viral genome reverse transcription is more complete, is adapted to the amplification of mycovirus rna genome, has wide
Application prospect.
Detailed description of the invention
Fig. 1 is the BLAST result of forward primer.
Fig. 2 is the dsRNA virus electrophoresis result figure in the present invention for genome amplification clone.Marker (5kbp), swimming
Road 1 and 2 is the dsRNA extracted in point spore anthrax bacteria.
Fig. 3 is that viral genome expands PCR product electrophoresis result in the present invention.Marker (5kbp), swimming lane 1 and 2 are PCR
Amplified production.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with the embodiment of the present invention, to this hair
Technical solution in bright embodiment is clearly and completely described, it is clear that described embodiment is that a part of the invention is implemented
Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creativeness
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of labour.
The sequence of known mycoviruses CaPV1 used in the following embodiment can be in US National Biotechnology Information
The heart (NCBI) retrieval obtains.
1 design of primers of embodiment
The present inventor searches for and the Iresine herbetii viroid Iresine of known viruse and fungi without homology from NCBI/GenBank
Viroid1 sequence (NC_003613.1).Using Primer.5 software design forward primer sequence and complementary primer sequences, wherein
5 ' ends of forward primer are phosphorylated modification, and 3 ' ends are modified by amination.
Forward primer: 5 '-PO4-CTGGAGCGAACTCGGCAAGG-NH2-3’
Complementary primer 2:5 '-CCTTGCCGAGTTCGCTCCAG-3 '
Above-mentioned primer is synthesized by precious bioengineering (Dalian) Co., Ltd.Forward primer is retrieved through BLAST, as a result sees figure
1, the results showed that identical with the covering of forward primer sequence 100% and 100% is entirely Iresine viroid1 sequence, and
Know that virus and fungal sequence do not have homology.
2 virus dsRNA of embodiment is extracted
The extraction of viral dsRNA refers to cellulose CF affinity chromatography, and operation key step is as follows:
(1) the mycelia 0.5-1g for taking fresh dried, with liquid nitrogen grinding at powder in mortar；
(2) 1 × STE buffer of 650 μ L, whirlpool vibration 3min is added；
(3) 4 DEG C, 10000g is centrifuged 1min；Supernatant is collected in new centrifuge tube；
(4) add isometric phenol: chloroform: isoamyl alcohol (25:24:1)；
(5) after whirlpool vibration 3min, 4 DEG C, 10000g is centrifuged 1min, and supernatant liquor is moved into new centrifuge tube；
(6) add the dehydrated alcohol of 50mg CF cellulose powder and 0.2 times of volume, whirlpool vibration 10min；
(7) 4 DEG C, 10000g is centrifuged 3min, abandons supernatant, collects powder；
(8) 1.2mL is added to contain 1 × STE mixed liquor of 15% ethyl alcohol, abundant whirlpool vibration 1min；
(9) 10000g is centrifuged 3min, collects powder, abandons supernatant；
(10) the sterile water elution dsRNA of 100 μ L, whirlpool vibration 1min is added；
(11) 4 DEG C, 10000g is centrifuged 3min, collects supernatant to another new centrifuge tube；
(12) step 10-11 step is repeated；Merge supernatant twice；
(13) (- 20 DEG C are protected the sodium acetate (pH5.2) and 2.5 times of cold dehydrated alcohols of volume that the 3.0M of 0.1 times of volume is added
Deposit), 60min is incubated for after mixing on ice；
(14) 4 DEG C, 15000g is centrifuged 10min, precipitates dsRNA, abandons supernatant；
(15) 1mL70% ethanol washing precipitating is added；
(16) 4 DEG C, 15000g is centrifuged 5min, abandons supernatant；
(17) it is dried in vacuo 3min；
(18) precipitating, -20 DEG C of short-term preservations or -80 DEG C of long-term preservations are dissolved with the TE of 20 μ L.
DsRNA extracts product through DNase I (RNase-free) and S1 Nuclease after 37 DEG C of processing 40min, use
1.0% Ago-Gel carries out electrophoresis, and electrophoresis 0.5h is placed on gel imaging system and observes and take pictures under 5V/cm constant-pressure conditions
It saves, obtains viral genome electrophorogram.Result is extracted as shown in Figure 1, size is in 1.5- containing dsRNA virus in bacterial strain
2kbp or so.
3 viral genomic clone of embodiment
(1) dsRNA is connect with forward primer
The dsRNA of 200ng is added in 20uL linked system, 10 × T4 RNA Ligase Buffer (contains 10mMATP)
2uL, PEG (50%) 10uL, T4 RNA Ligase 0.5uL, T4 DNA Ligase 0.5uL, BSA (0.1%) 1uL,
Primer 1 (20uM) 1uL, finally uses ddH2O complements to 20uL.
Reaction condition are as follows: 37 DEG C of connections 3h, 16 DEG C of connection 16h.DNTP 0.5uL, Taq DNA are added in connection product
Polymerse 0.5uL and 10 × PCR Buffer 3uL, ddH2O complements to 30uL, 70 DEG C of reaction 20min.
(2) synthesis of the first chain of reverse transcription and cDNA
Using the dsRNA of forward primer connection as template, reverse transcription is carried out using complementary primer and random primer and synthesizes cDNA
First chain.Reaction system is as follows: connection product 4ul, and complementary primer 2uL and DMSO 2uL are added the processed 2uL of DEPC and are centrifuged
Guan Zhong is immediately placed in 1min in liquid nitrogen, rear dislocation is on ice in 99 DEG C of water-bath 3min；It sequentially adds, 5 × RT Buffer
4uL, dNTP 2uL, DTT 2uL, RNase Inhibitor 1uL, SuperScriptTM II RNase H-Reverse
Transcriptase1uL, 42 DEG C of reaction 1h.
(3) synthesis of the second chain of cDNA
Taking the cDNA of synthesis is template, carries out PCR amplification with complementary primer.PCR reaction system includes: ddH2O
34.5uL, 10 × PCR Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 1uL, cDNA 10ul,
LA TaqTM 0.5uL.Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 2min 35 are recycled, so totally
72 DEG C of extension 5min afterwards.
(4) after reaction terminating, 5uL PCR product is taken, 1% agarose electrophoresis detects amplification, electrophoresis result such as Fig. 2 institute
Show.
By amplified fragments gel extraction, Escherichia coli (Escherichia coli) DH5 α is ligated and transformed into carrier T.Bacterium
Picking positive colony is sequenced after falling PCR detection, obtains virus genome sequence.Sequence is subjected to BLAST comparison.
With forward primer and complementary primer of the invention to the Colletotrichum acutatum of double split Viraceae
Partitivirus1 (CaPV1) carries out whole genome amplification and cloning and sequencing, the available virus full length sequence.
Above embodiments explanation is complete using adapter-primer 1 of the invention and the available rna virus cdna group of complementary primer
Long sequence.
Sequence table
<110>Agricultural University Of Hunan
<120>a kind of primer and method for unknown RNA mycoviruses genomic clone
<141> 2019-05-07
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
ctggagcgaa ctcggcaagg 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
ccttgccgag ttcgctccag 20
Claims (8)
1. a kind of primer for unknown RNA mycoviruses genomic clone, which is characterized in that the primer includes forward primer
And complementary primer, the nucleotide sequence of the forward primer is as shown in SEQ ID No.1, the nucleotide sequence of the complementary primer
As shown in SEQ ID No.2.
2. a kind of primer for unknown RNA mycoviruses genomic clone according to claim 1, which is characterized in that
The forward primer and known viruse sequence are without homology, and 5 ' hold phosphorylations, 3 ' Amino End Groups.
3. a kind of primer for unknown RNA mycoviruses genomic clone according to claim 1, which is characterized in that
The unknown RNA mycoviruses include diplornavirus and single strand RNA virus.
4. a kind of method for carrying out unknown RNA mycoviruses genomic clone using primer described in claim 1, feature exist
In, comprising the following steps:
(1) extraction of mycoviruses dsRNA；
(2) forward primer is attached using ligase with dsRNA, and connection product is purified；
(3) using the connection product as template, reverse transcription is carried out using the complementary primer and random primer and synthesizes cDNA first
Chain；
(4) using first chain of cDNA as template, PCR amplification is carried out with the complementary primer, obtains pcr amplification product；
(5) cloning and sequencing is carried out to pcr amplification product.
A kind of primer described in claim 1 is utilized to carry out unknown RNA mycoviruses genome 5. according to claim 4
The method of clone, which is characterized in that the linked system in the step (2) is specific as follows: being added in 20uL linked system
The dsRNA of 200ng, 10 × T4 RNA Ligase Buffer (containing 10mMATP) 2uL, PEG (50%) 10uL, T4 RNA
1 (20uM) 1uL of Ligase 0.5uL, T4 DNA Ligase 0.5uL, BSA (0.1%) 1uL, Primer, finally uses ddH2O
Complement to 20uL；
Reaction condition are as follows: dNTP 0.5uL, Taq DNA is added in 37 DEG C of connections 3h, 16 DEG C of connection 16h in connection product
Polymerse 0.5uL and 10 × PCR Buffer3uL, ddH2O complements to 30uL, 70 DEG C of reaction 20min.
A kind of primer described in claim 1 is utilized to carry out unknown RNA mycoviruses genome 6. according to claim 4
The method of clone, which is characterized in that the reaction system that the first chain of cDNA synthesizes in the step (3) is as follows: connection product 4ul,
Complementary primer 2uL and DMSO 2uL is added in the processed 2uL centrifuge tube of DEPC, in 99 DEG C of water-bath 3min, is immediately placed in liquid nitrogen
Middle l min, rear dislocation is on ice；It sequentially adds, 5 × RT Buffer 4uL, dNTP 2uL, DTT 2uL, RNase
Inhibitor 1uL, SuperScriptTM II RNase H-Reverse Transcriptase 1uL, 42 DEG C of reaction 1h.
A kind of primer described in claim 1 is utilized to carry out unknown RNA mycoviruses genome 7. according to claim 4
The method of clone, which is characterized in that PCR reaction system includes: ddH in the step (4)2O 34.5uL, 10 × PCR
Buffer 5uL, dNTP Mixture (2.5mM) 6uL, Primers (20mM) 1uL, cDNA 10ul, LA TaqTM 0.5uL；
Amplification condition are as follows: after 94 DEG C of 1min, 94 DEG C of 10sec, 56 DEG C of 10sec, 72 DEG C of 2min, totally 35 circulation, then extends for 72 DEG C
5min。
A kind of primer described in claim 1 is utilized to carry out unknown RNA mycoviruses genome 8. according to claim 4
The method of clone, which is characterized in that this method is for obtaining unknown virus dsRNA full length cDNA sequence.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111635930A (en) * | 2020-05-12 | 2020-09-08 | 中国农业科学院农业基因组研究所 | Method for high-throughput sequencing and calling full-length sequence of unknown RNA |
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| CN101864435A (en) * | 2010-04-27 | 2010-10-20 | 中国科学院水生生物研究所 | Turbot reovirus whole genome and application thereof |
| CN102226195A (en) * | 2011-05-12 | 2011-10-26 | 湖南农业大学 | Method for cloning viroid complete-genome sequence |
| CN106676096A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院植物研究所 | Whole set of reagents and method for cloning full-length gene |
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| CN101864435A (en) * | 2010-04-27 | 2010-10-20 | 中国科学院水生生物研究所 | Turbot reovirus whole genome and application thereof |
| CN102226195A (en) * | 2011-05-12 | 2011-10-26 | 湖南农业大学 | Method for cloning viroid complete-genome sequence |
| CN106676096A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院植物研究所 | Whole set of reagents and method for cloning full-length gene |
Non-Patent Citations (1)
| Title |
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| SPIEKER,R.L.: "NCBI Reference Sequence: NC_003613.1", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635930A (en) * | 2020-05-12 | 2020-09-08 | 中国农业科学院农业基因组研究所 | Method for high-throughput sequencing and calling full-length sequence of unknown RNA |
| CN111635930B (en) * | 2020-05-12 | 2023-10-24 | 中国农业科学院农业基因组研究所 | Method for extracting unknown RNA full-length sequence by high-throughput sequencing |
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