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Construction methods and applications of PVX (potato virus X) over-expression vectors and BiFC (bimolecular fluorescence complementation) vector

A construction method, potato technology, applied in the field of genetic engineering

Inactive Publication Date: 2014-08-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, vectors such as pGR106 can only express one protein

Method used

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  • Construction methods and applications of PVX (potato virus X) over-expression vectors and BiFC (bimolecular fluorescence complementation) vector
  • Construction methods and applications of PVX (potato virus X) over-expression vectors and BiFC (bimolecular fluorescence complementation) vector
  • Construction methods and applications of PVX (potato virus X) over-expression vectors and BiFC (bimolecular fluorescence complementation) vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Extraction of plant total RNA

[0037] Take about 0.1g of the upper leaves of PVX-infected tobacco plants, grind them thoroughly in liquid nitrogen, add 1mL TRIZOL, vortex, incubate at room temperature for 5min, add chloroform, vigorously vortex for 15s, incubate at room temperature for 3min, and centrifuge at 13000r / min for 15min. Carefully draw the supernatant, add 1 / 2 volume of isopropanol, let stand for 10 minutes, and centrifuge at 13000r / min for 10 minutes. The precipitate was washed with 75% ethanol and centrifuged at 13000r / min for 5min. The precipitate was redissolved with 50 μL DEPC water and stored at -80°C for later use.

Embodiment 2

[0038] Example 2: Construction of Potato Virus X Infectious Clones

[0039] Using the viral RNA obtained in Example 1 as a template, reverse transcription was performed with random primers. According to the existing restriction enzyme map of the entire genome of potato virus X, it can be amplified in three parts, and assembled into a PVX full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the fragment PVX 5'untranslated region (UTR) to the upstream of the nt543-BamHI restriction site by Overlap-PCR, and the inventor named the fragment p35S-543; PCR amplified 543- The fragment between the BamHI restriction site and the 3349-EcoRI restriction site was named by the inventor as fragment 543-3349; PCR amplification of the 3349-EcoRI restriction site to the poly(A) tail Fragment, the inventor named the fragment 3349-polyA.

[0040] Using total plant RNA as a template, Moloney murine leukaemia virus reversetranscriptase (Promega) was used for reverse...

Embodiment 3

[0048] Embodiment 3: the preparation of potato X virus overexpression vector

[0049] Primer 11 and primer 12 amplify the CP promoter 209bp of TMV plasmid, primer 7 (with ApaI restriction site) and primer 14 amplify a 599bp band, primer 13 and primer 8 (with SmaI restriction site) The 864bp band was amplified, the 3 bands obtained above were recovered, mixed, amplified with primer 7 and primer 8, and the TMV CP promoter was inserted between the PVX CP promoters by Overlap-PCR, and in A multiple cloning site (AsisI, SacI, BstBI, MluI) was inserted before and after the TMV CP promoter. ApaI upstream of the TGB gene and SmaI downstream of the 3'-UTR were double-digested and inserted between the corresponding restriction sites of pCaPVX100 to construct a vector capable of simultaneously expressing two foreign genes. The expression vector was named pCaPVX440 ( figure 1 ), the sequence is shown in Seq ID No:32.

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Abstract

The invention relates to the field of genetic engineering and provides construction methods and applications of two PVX (potato virus X) over-expression vectors and one BiFC (bimolecular fluorescence complementation) vector. A genome of a PVX 1985 isolate is cloned to a downstream 35S promoter through genetic recombination, obtained infectious clone can infect solanaceae crops such as tobacco, tomatoes, potatoes and the like through agrobacterium infiltration. Infectious clone is reconstructed, and the over-expression vectors capable of effectively expressing one or two types of heterologous protein in a host plant body as well as the BiFC vector capable of identifying interactions between protein are obtained.

Description

technical field [0001] The present invention relates to the field of genetic engineering, specifically, the present invention relates to the construction method and application of the invasive clone of Potato virus X (PVX) 1985 isolate, an overexpression vector and a bimolecular fluorescent complementary vector of the genus Potatovirus . Background technique [0002] Infectious cloning is an important tool for studying the function of plant viral genomes. Using infectious clones of viruses, overexpression vectors can also be constructed. Compared with transgenic technology, the technology of using overexpression vectors to express foreign proteins in plants has the advantages of simple operation, low cost, short production cycle and high expression level. [0003] Bimolecular fluorescence complementation (BiFC) assay is an important method for identifying protein interactions. The main principle is that the gene fragments encoding the N and C segments of yellow fluorescen...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/66G01N33/68
Inventor 李向东王莹丛倩倩
Owner SHANDONG AGRICULTURAL UNIVERSITY
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