Primer group and method for cloning whole genome of sugarcane isolate with sorghum mosaic virus
A sorghum mosaic virus and whole genome technology, which is applied in the directions of recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of long time consumption, cumbersome experimental steps, difficulty in obtaining a full-length genome sequence, etc., and the method is simple and convenient. Effect
Inactive Publication Date: 2013-09-18
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology
Since SrMV often infects sugarcane mixed with SCMV or SCSMV, it is extremely difficult to isolate and purify, and it takes a long time. The experimental steps are very cumbersome and require expensive ultracentrifuges
RT-PCR cloning by extracting total RNA from susceptible plant leaves is a simple method, but because the genomic RNA sequence is about 10 kb long, it is difficult to obtain the full-length genome sequence by 1 RT-PCR cloning
Information about SrMV genome is very limited at home and abroad
Method used
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Embodiment 1
[0043] Embodiment 1 A kind of method for cloning the whole genome of sorghum mosaic virus sugarcane isolate comprises the following steps:
[0044] 1. Extraction of total RNA from sugarcane leaves
[0045] 1) 16 field sugarcane leaf samples were collected from sugarcane areas in various provinces (autonomous regions) of my country, and stored at -80 ℃ for future use.
[0046] 2) Weigh 0.1 g of sugarcane leaves and grind them into powder with liquid nitrogen.
[0047] 3) Add the powder to a 1.5 mL centrifuge tube, and add 1 mL immediately after the liquid nitrogen has just evaporated Reagents, capped the tube, vortexed to mix, and centrifuged at 12000 g for 10 min at 4 °C.
[0048] 4) Pipette the supernatant into another new centrifuge tube, add 0.2 mL of chloroform, cover the cap of the tube, vortex and mix well, let stand at room temperature for 3 min, and then centrifuge at 12000 g for 15 min at 4 °C.
[0049] 5) Pipette the supernatant into another new centrifuge tube, ...
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The invention relates to a primer group and method for cloning a whole genome of a sugarcane isolate with a sorghum mosaic virus. The primer group for cloning the whole genome of the sugarcane isolate with the sorghum mosaic virus comprises seven pairs of primers which sequentially have the nucleotide sequences shown as SEQ ID NO.1 to SEQ ID NO.14 in a sequence table; and a total-length sequence of an SrMV genome is obtained through carrying out inverse transcription reaction by taking the total RNA (Ribose Nucleic acid) of an SrMV infected sugarcane lamina as a template, carrying out PCR (Polymerase Chain Reaction) amplification by using the primers by taking an inverse transcription product as a template and sequencing and splicing all fragments. Primer amplified fragments of the primer group are overlapped with each other, the approximate total-length sequence of the SrMV genome is covered, and the whole-genome sequences of different SrMV strains can be cloned; and the invention provides a method for cloning the whole SrMV genome in sections, and the method has the advantages of simplicity, convenience, effectiveness and rapidness and provides a convenient, rapid and effective new approach for cloning the whole SrMV genome sequence.
Description
technical field [0001] The invention relates to a primer set and a method for cloning the whole genome of a plant virus, in particular to a primer set and a method for cloning the whole genome of a sorghum mosaic virus sugarcane isolate, and belongs to the field of biotechnology. Background technique [0002] Sorghum mosaic virus ( Sorghum mosaic virus , SrMV) is a typical member of the genus Potyvirus in the family Potyviridae. The virion is rod-shaped, with a size of 620×15 nm. The genome is a single, positive-strand RNA, about 10 kb in length. The 5′ end is connected to the genome-binding protein, and the 3′ end has a poly (A) sequence. The genome encodes a polyprotein, which is then degraded by its own encoded protease to form 10 polypeptides, namely P1, HC-Pro, P3, 6 K1, CI, 6 K2, VPg, NIa, NIb and CP. SrMV can be associated with Potatovirus Y sugarcane mosaic virus ( Sugarcane mosaic virus , SCMV) and Poacevirus Sugarcane stripe mosaic virus (SCMV) Sugarcane st...
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Login to View More IPC IPC(8): C12N15/11C12N15/10
Inventor 高三基傅华英王英余永东陈如凯孙生仁陈复资
Owner FUJIAN AGRI & FORESTRY UNIV