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Cloning and transcription carrier construction of cymbidium mosaic virus strain

A technology for building orchid leaves and virus strains, applied in the field of molecular biology, can solve the problems of inability to obtain virus sequences, inability to obtain subgenomes, and ignoring the existence of quasi-species of virus strains

Inactive Publication Date: 2019-06-07
SHANGHAI CHENSHAN BOTANICAL GARDEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, primers could only be designed based on known sequences, and sequences could be obtained by segmented amplification or full-length amplification. This method could only obtain sequences similar to known sequences and was not specific; The method of amplifying virus strains and re-splicing ignores the fact that quasi-species of virus strains exist in the natural state, and cannot obtain the real virus sequence; the method of full-length amplification is often designed according to the sequence of the 5' end and 3' end of the genome Primers, the situation of the subgenome cannot be obtained; and for the CymMV subgenome, there are only three subgenomes at the 3' end, and there is no report on the 5' end subgenome

Method used

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  • Cloning and transcription carrier construction of cymbidium mosaic virus strain
  • Cloning and transcription carrier construction of cymbidium mosaic virus strain
  • Cloning and transcription carrier construction of cymbidium mosaic virus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1-Determination of the sequence of the orchid mosaic virus strain

[0051] This embodiment is to establish the determination of the orchid mosaic virus strain sequence, which includes the following aspects:

[0052] (1) Processing of high-throughput data;

[0053] Using Bowetie2 to compare the high-throughput data to 14 known full-length genome sequences of Jianlan mosaic virus, and using genomeCoverageBed to calculate the read coverage (ReadCoverage) of each position in the Jianlan mosaic virus genome. The 14 sequences were aligned at the alignment position, and the Read Coverage of the position where the sequence was missing was recorded as 0. Use the Plot function in R to make graphs, see figure 1 .

[0054] Each sequence is abbreviated as CymMV, Cymbidium mosaic virus—Taiwan[AY571289.1]; Nanjing[JQ860108.1]; Korea[AF016914.1]; China[KR185347.1]; Kunming [AM055640.2]; ]; India[AM055720.1]; Singapore[U62963.1]; Japan[AB197937.1]; TaiwanM2[EU314803.1]; J...

Embodiment 2

[0071] Construction of embodiment 2-infectious cDNA vector

[0072] This embodiment is the construction of the in vitro transcription vector of the full-length genome sequence of CymMV and the 5' end subgenome of CymMV in Example 1, which adopts the following primers:

[0073] The sequence of the forward primer introduced into the T7 promoter is:

[0074] aaaacgacggccagtgaattcATAATACGACTCACTATAGGGAGAGGAA (SEQ ID NO: 22)

[0075] The reverse primer introduces the PolyA site and the SmaI blunt end restriction site:

[0076] gcctctgcagtcgacggggcccACCCGGGTTTTTTTTTTTTTTTT (SEQ ID NO: 23)

[0077] The full-length and subgenome fragments of CymMV were respectively connected to the vector pUC57, and the sequences of the in vitro transcription vectors of the full-length genome sequence of CymMV and the 5' end subgenome of CymMV were respectively shown in SEQ ID NO: 24 and SEQ ID NO: 25, Its structural diagram is as image 3 As shown, the constructed in vitro transcription vector co...

Embodiment 3

[0078] Preparation and infection verification of embodiment 3-CymMV virus RNA

[0079] This embodiment uses the in vitro transcription vector constructed in Example 2 to carry out the preparation and infection verification of CymMV virus RNA. The specific steps include:

[0080] (1) Linearization of in vitro transcription vectors;

[0081] After the vector was digested with restriction endonuclease SmaI, it was detected by agarose gel electrophoresis to ensure that the vector was completely linearized.

[0082] (2) Synthesis of in vitro transcripts;

[0083] Add 2μL of 10× in vitro transcription buffer, 2μL of 100mM ATP, 2μL of 100mM CTP, 2μL of 100mM UTP, 2μL of 20mM GTP, 4μL of 40mM m7G(5')ppp(5')G, and 4μL of linearized template in a 1.5mL EP tube , T7 RNA polymerase (NEB company) 2uL, 37 ° C water bath for 2 hours, agarose electrophoresis to detect the quality and concentration of transcripts.

[0084] (3) Inoculation of in vitro transcripts;

[0085] The transcribed R...

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Abstract

The invention discloses a cymbidium mosaic virus strain. The sequence of an overall length genome is shown as SEQID NO:1-10, and the sequence of 5' terminal subgenome is shown as SEQID NO:11-21. The invention further discloses an in vitro transcription carrier of an overall length genome sequence and / or 5' terminal subgenome, a primer compound for the overall length genome sequence and / or 5' terminal subgenome and an amplifying method, and a method for preparing cymbidium mosaic viruses by using the in vitro transcription carrier. The natural state that virus strain quasispecies exists is considered, according to the overall length genome sequence and the 5' terminal subgenome of the cymbidium mosaic virus strain prepared by the method disclosed by the invention, the constructed carrier invitro transcription substance has infection activity, not only can increase the abundance of cymbidium mosaic virus genome sequence, but also can further deeply establish the foundation for researchof the gene recombination, the genome function and the infective molecular mechanism of the Cymbidium mosaic viruses.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a Jianlan mosaic virus strain, in particular to the full-length genome sequence and 5' end subgenome sequence of the virus, its in vitro transcription vector, its amplification primers and amplification method and The method of preparation of the virus. Background technique [0002] Cymbidium mosaic virus (CymMV) is a member of the order Turnipymoviridae, the family Alphaviridae, and the genus Potatovirus. Potato virus X (PVX) is the model virus of the same genus. The virion is composed of coat protein and positive-sense single-stranded RNA genome (+ssRNA). It is one of the viruses with the most extensive hosts and the most serious damage in Orchidaceae. Orchid and other tropical orchids and national orchids. Orchid mosaic virus can infect orchids alone or in combination with Odontoglossum ringspot virus (ORSV), causing symptoms such as chlorotic stripes, depressed gray...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/82C12N15/11C12N15/70
Inventor 杨洪星王令金恩由董海鸿张霞庄海燕
Owner SHANGHAI CHENSHAN BOTANICAL GARDEN
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