The primers and cloning method for cloning the full-length genome sequence of sweet potato feather mottle virus o strain and sweet potato virus c

A feathery mottle virus and genome sequence technology, applied in the field of bioengineering, can solve the problems of low content, difficult to obtain the full-length genome sequence of the virus, and no full-length genome sequence of the isolates yet.

Active Publication Date: 2017-08-04
INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since SPFMV and SPVC often infect sweet potatoes in combination, separation and purification are extremely difficult, and the content of the two viruses in sweet potatoes is low, so it is difficult to obtain the full-length genome sequence of the virus by conventional cloning methods of purified viruses
So far, there have been no reports of the full-length genome sequences of SPFMV and SPVC Chinese isolates

Method used

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  • The primers and cloning method for cloning the full-length genome sequence of sweet potato feather mottle virus o strain and sweet potato virus c
  • The primers and cloning method for cloning the full-length genome sequence of sweet potato feather mottle virus o strain and sweet potato virus c
  • The primers and cloning method for cloning the full-length genome sequence of sweet potato feather mottle virus o strain and sweet potato virus c

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 A kind of method for cloning the full-length genome sequence of SPFMV-O strain

[0066] 1. Materials and methods

[0067] (1) Tested virus material: the leaves of sweet potato plants infected with SPFMV, SPVC and SPCSV were used as materials. The symptoms were as follows: the plants were short, the leaves were shrunken, yellowed, and the veins were distorted. The total RNA was extracted for subsequent experiments.

[0068] (2) Reagents and kits: UNIQ-10 Column Total RNA Extraction Kit was produced by Shanghai Sangon Bioengineering Company; DNA Gel Recovery Kit was purchased from Axygen Biotechnology (Hangzhou) Co., Ltd.; RNase inhibitor, AMV Reverse transcriptase, LATaq DNA polymerase, and pMD19-T vector were purchased from TaKaRa Company, and other commonly used reagents were of domestic analytical grade.

[0069] (3) Primer design: According to the nucleotide sequence of the full-length genome of SPFMV-O registered in GenBank (accession numbers are AB509...

Embodiment 2

[0088] Example 2 A method for cloning the full-length genome sequence of sweetpotato virus C

[0089] 1. Materials and methods

[0090] (1) Tested virus material: the leaves of sweet potato plants infected with SPFMV, SPVC and SPCSV were used as materials. The symptoms were as follows: the plants were short, the leaves were shrunken, yellowed, and the veins were distorted. The total RNA was extracted for subsequent experiments.

[0091] (2) Reagents and kits: UNIQ-10 Column Total RNA Extraction Kit was produced by Shanghai Sangon Bioengineering Company; DNA Gel Recovery Kit was purchased from Axygen Biotechnology (Hangzhou) Co., Ltd.; RNase inhibitor, AMV Reverse transcriptase, LATaq DNA polymerase, and pMD19-T vector were purchased from TaKaRa Company, and other commonly used reagents were of domestic analytical grade.

[0092] (3) Primer design: According to the nucleotide sequence of the full-length genome of SPVC registered in GenBank (accession numbers are JX489166, AB50...

Embodiment 3

[0107] Example 3 A method for simultaneous cloning of sweet potato feather mottle virus O strain and sweet potato virus C full-length genome sequence

[0108] 1. Materials and methods

[0109] (1) Tested virus material: the leaves of sweet potato plants infected with SPFMV, SPVC and SPCSV were used as materials. The symptoms were as follows: the plants were short, the leaves were shrunken, yellowed, and the veins were distorted. The total RNA was extracted for subsequent experiments.

[0110] (2) Reagents and kits: UNIQ-10 Column Total RNA Extraction Kit was produced by Shanghai Sangon Bioengineering Company; DNA Gel Recovery Kit was purchased from Axygen Biotechnology (Hangzhou) Co., Ltd.; RNase inhibitor, AMV Reverse transcriptase, LATaq DNA polymerase, and pMD19-T vector were purchased from TaKaRa Company, and other commonly used reagents were of domestic analytical grade.

[0111] (3) Primer design: according to the nucleotide sequences of the full-length genomes of SPFMV...

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Abstract

The invention discloses primers for cloning SPFMV (sweet potato feathery mottle virus) O strain and full length genome sequence of SPVC (sweet potato virus C). The primers are respectively a primer for cloning the SPFMV O strain, a primer for cloning the full length genome sequence of SPVC, and the primer for simultaneously cloning the SPFMV O strain and the full length genome sequence of SPVC. The SPFMV and the SPVC generally infect sweet potatoes in a mixed way, the separation and the purification are very difficult; the content of the virus in the sweet potatoes is low; the full length genome sequence is difficult to obtain by a conventional virus purification cloning method. The principle that SPCSV can promote the content rise of the SPFMV and the SPVC in the sweet potato plants is used; the sweet potato plants simultaneously infected with SPCSV, SPFMV-O and SPVC viruses are used as materials; the total RNA (ribonucleic acid) of the virus-infecting sweet potato plant leaves is extracted; the virus full length genome sequence cloning is performed; through a great amount of PCR (polymerase chain reaction) amplification and sequence testing, the genome full length sequence of SPVC and SPFMV is obtained through comparison and splicing.

Description

technical field [0001] The invention relates to primers and a cloning method for cloning the full-length genome sequence of sweet potato feather mottle virus O strain, primers and a cloning method for cloning the full-length genome sequence of sweet potato virus C, and simultaneous cloning of sweet potato feather mottle virus O strain and sweet potato virus C A primer and a cloning method for a full-length genome sequence belong to the technical field of bioengineering. Background technique [0002] Sweet potato feathery mottle virus (Sweet potato feathery mottle virus, SPFMV) and sweet potato virus C (Sweet potato virus C, SPVC) are the main viruses that harm sweet potatoes, and both viruses belong to the family Potyviridae (Potyviridae). Potyvirus) members, often mixed infecting sweet potatoes in the field, and can co-grow with sweet potato chlorotic stunt virus (SPCSV) to cause the occurrence of sweet potato compound virus disease SPVD (sweet potato virus disease, SPVD), ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12N15/40
CPCC07K14/005C12N15/10C12N15/11C12N2770/34022C12Q2531/113
Inventor 秦艳红张振臣乔奇张德胜王永江田雨婷王爽渠瑞娜
Owner INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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