Construction and application of tobacco vein banding mosaic virus infectious clone
A Mosaic Virus, Vein Banding Technology
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Embodiment 1
[0037] Example 1: Purification of Tobacco Vein Mosaic Virus Particles
[0038] About 200 g of diseased leaves were weighed and left overnight at 4°C. Add 200mL of 0.2mol / L phosphate buffer (pH=8.0, 0.15% mercaptoethanol, 0.01mol / L EDTA) to homogenate, and centrifuge at 8000r / min at 4°C for 15-20 minutes. Add 1% TritonX-100 to the supernatant and stir at 4°C for 3 hours, add PEG600040g / L and NaCl (0.2M final concentration) and stir for 1.5 hours, then centrifuge at 8000r / min at 4°C for 15-20 minutes. Precipitate with 0.2mol / L phosphate buffer (pH=8.0, 1%TritonX-100) 1 / 10 of the original volume and gently shake overnight at 4°C. Centrifuge at low speed to remove insoluble impurities. The supernatant was centrifuged at 40,000 r / min for 1 hour, and the pellet was resuspended with 8 mL of 0.2 mol / L phosphate buffer, and kept overnight at 4°C. Carefully place 2mL of the virus suspension on the top layer of 30% sucrose (prepared in 0.05M phosphate buffer), and centrifuge at 90,000...
Embodiment 2
[0039] Embodiment 2: the extraction of virus RNA
[0040] Take 200 μL of virus extract and add an equal volume of extraction buffer (40mmol / L Tris-Cl, pH9.0, 2mmol / L EDTA, 2% SDS), vortex, add RNase inhibitor, 65°C for 5 minutes. Add an equal volume of phenol, vortex, 60°C for 1 minute, and centrifuge at 13000r / min for 5 minutes. Add an equal volume of phenol / chloroform (1:1, pH=5.2) to the supernatant, and centrifuge at 13000r / min for 5 minutes. The supernatant was precipitated with absolute ethanol and centrifuged at 13000r / min for 5 minutes. The precipitate was redissolved with 50 μL DEPC water and stored at -80°C for later use.
Embodiment 3
[0041] Example 3: Construction of Tobacco Vein Mosaic Virus Infectious Clones
[0042] Using the viral RNA obtained in Example 2 as a template, reverse transcription was performed with random primers. According to the existing restriction enzyme map of the whole genome of tobacco vein mosaic virus, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5' untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplified HC- The fragment between the pro gene Nru I restriction site and the 6K2Xho I restriction site, the inventor named the fragment pHC-6K2; PCR amplification of the 6K2Xho I restriction site to the poly(A) tail Fragment, the inventor named the fragment p6K2-polyA ( figure 1 ).
[0043] The reverse transcriptase used for cDNA synthesis was Moloney m...
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