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Methods of Proliferating Functional Potato y Viruses in Prokaryotic Cells

A technology of prokaryotic cells and potatoes, applied in the biological field, can solve the problems of increasing the potential for virus sequence mutation, non-viral nucleotides, affecting virus infection, transcription, and the inability of viruses to successfully construct invasive clones, etc., to improve Intrinsic Toxicity, Effect of Yield Improvement

Active Publication Date: 2022-08-02
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many viruses in this genus that cannot successfully construct invasive clones
In addition, traditional methods for constructing invasive clones involve multiple subcloning and enzyme-ligating steps, which increase the potential for mutations in the viral sequence and the introduction of non-viral nucleotides, thereby affecting viral survival. Infection, transcription and other biological characteristics

Method used

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  • Methods of Proliferating Functional Potato y Viruses in Prokaryotic Cells
  • Methods of Proliferating Functional Potato y Viruses in Prokaryotic Cells
  • Methods of Proliferating Functional Potato y Viruses in Prokaryotic Cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction Strategy

[0063] Reported PVY NTN-NW The strain isolate SYR-II-2-8 (GenBank accession number: AB461451) has a prokaryotic promoter-like element in the P3 gene ( figure 1 , bold and underlined), according to this result in PVY NTN-NW A prokaryotic promoter-like element was also localized in JL-W1 of the strain isolate ( figure 1 , bold underline mark), in the middle of the element ( figure 1 , the bold italic mark) was inserted into intron A (IntronA / ST-LS1 / S. tuberosum) of SEQ ID NO.2 to disrupt its structure, thereby blocking the expression of viral proteins in E. coli.

Embodiment 2

[0064] Example 2 Amplification and ligation of fragments of invasive clones

[0065] Using the JL-W1 isolate cDNA, potato DNA and plasmid containing cauliflower mosaic virus (CaMV) 35S promoter preserved in the laboratory as templates, the virus fragment, intron and vector fragment were amplified respectively. A 50 μL reaction system was used for PCR amplification: PrimeSTAR HS (Premix) 25 μL, template 1 μL, forward primer (10 μmol L-1) 1 μL, reverse primer (10 μmol L-1) 1 μL, and ultrapure water 22 μL. PCR conditions: denaturation at 98°C for 10s, annealing for 10s (annealing temperature is shown in Table 1), extension at 72°C for several minutes (the extension time is calculated as 1 kb / min), a total of 30 cycles. After the reaction, PCR products were detected by 1.5% agarose gel electrophoresis. After detection, the target band was cut out and purified using a gel recovery kit, and the purified product was stored at -20°C.

[0066] The primers used to construct and verify...

Embodiment 3

[0076] Example 3 Identification of recombinant plasmids

[0077] (1) Enzyme digestion identification: 5 μL of each extracted plasmid was taken, detected by 1% agarose gel electrophoresis, and the plasmids meeting the expected size were screened. Afterwards, the restriction endonucleases BamHI and PstI were selected, and the recombinant plasmids meeting the expected size were respectively subjected to single-enzyme digestion verification. After the reaction, 1% agarose gel electrophoresis was used to detect the result of restriction digestion.

[0078] Pick a single clone colony for plasmid extraction, and the extraction results are analyzed by agarose gel electrophoresis, such as Figure 4 As shown (M: DNA molecular standard; 1-12: plasmid Z1-12), the plasmids were all in line with the expected size.

[0079] The positive recombinant plasmids screened by BamHI and PstI were subjected to single-enzyme digestion verification using restriction endonucleases, and the verificatio...

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Abstract

The present invention relates to the field of biotechnology, and in particular, provides a method for proliferating functional potato Y virus in prokaryotic cells. In the present invention, an intron is inserted into the prokaryotic promoter region of the potato Y virus cDNA to reduce or eliminate the promoter activity of the prokaryotic promoter region. the same amino acid sequence. This solution improves the inherent toxicity of the PotY virus cDNA to prokaryotic cells in the prior art, avoids the impact of PotY virus on the growth of prokaryotic cells, and improves the yield of the PotY virus cDNA without changing the infection of the PotY virus RNA transcript. sex.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to a method for proliferating functional potato Y virus in prokaryotic cells. Background technique [0002] Potato virus Y (PVY) is one of the most serious plant viruses, and it is widely distributed in potato and tobacco growing areas around the world. After PVY infects potatoes and tobacco, it will cause symptoms such as leaf vein necrosis, leaf yellowing and chlorosis, tuber ring spot necrosis, etc., resulting in the degradation of potato and tobacco germplasm, and the reduction of yield, causing serious losses to potato and tobacco production. [0003] PVY is a representative member of the genus Potyvirus of the family Potyviridae. The virion is curved and linear. Its genome is composed of positive-sense single-stranded RNA, about 9.7kb long, and contains a large open reading frame (Open reading frame). reading frame, ORF), both ends contain untranslated regions (UTRs). D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N7/00C12N15/70C12N1/21C12R1/94C12R1/19
CPCC12N7/00C12N15/70C12N2770/34021C12N2770/34052
Inventor 王永志苏颖李小宇张春雨马俊丰
Owner JILIN ACAD OF AGRI SCI
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