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Construction method of infectivity clones for black nightshade curtoviruses

A technology of virus infectivity and construction method, applied in the field of constructing infectious clones of Solanum nigrum virus Positive control of plant materials and other issues to achieve high fidelity, simple method, and high pathogenicity

Inactive Publication Date: 2020-03-24
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the current industry, there are no reports of Solanum solanum virus disease in my country
After reviewing the literature, no research related to Solanum nigrum virus has been found, and there is a lack of reports on the construction of a reverse genetics operating system for Solanum nigrum virus; in terms of virus detection, there is also a lack of positive control plant materials for Solanum nigrum virus, resulting in related Virology research cannot be carried out

Method used

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  • Construction method of infectivity clones for black nightshade curtoviruses
  • Construction method of infectivity clones for black nightshade curtoviruses
  • Construction method of infectivity clones for black nightshade curtoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Determination of the whole genome sequence of Solanum nigrum virus

[0034] (1) Plant DNA Extraction

[0035] Use the CTAB method to extract the total DNA of virus-infected tissues. The specific method is: take 0.2g of plant tissue leaves, grind them into powder in liquid nitrogen, transfer the powder to a 1.5ml centrifuge tube, and add 600 μL of CTAB preheated at 65°C Quickly mix the extract, keep it warm at 65°C for 30 minutes, and mix it upside down from time to time; after cooling to room temperature, add 500 μL of chloroform / isoamyl alcohol (24:1), mix upside down, and centrifuge at 12,000 rpm for 5 minutes; take the supernatant , add 600 μL of pre-cooled isopropanol, invert and mix well, centrifuge at 12000 rpm for 5 min; discard the supernatant, and wash the precipitate twice with 800 μL, 70% ethanol; slightly dry, dissolve the DNA precipitate in 30 μL of sterilized Store in double distilled water at 4°C.

[0036] (2) Amplification of viral sequenc...

Embodiment 2

[0066] Embodiment 2: the construction of Solanum solanum nigrum virus invasive clone

[0067] See the build process figure 1 , Geminivirus infectious cloning requires 1.3–2.0 full-length copies of the virus (need to carry two viral gene spacer sequences, ie IR regions). The pCB301 vector was used as a template, and P1 and P2 were used as primers to carry out PCR, and the product was used as the vector backbone of NCTV invasive clone. Using pLB-NCTV as a template and P3 and P4 as primers, PCR amplifies the first copy fragment of NCTV (length 2867bp). Using pLB-NCTV as a template and P5 and P6 as primers, PCR amplifies the second copy fragment of NCTV (length 2867bp). Through primer design, ensure that the above vector and the two copy fragments contain 15 bp homologous sequences, and use the multi-fragment homologous recombination cloning technology (Infusion) to obtain an invasive cloning vector containing two copies of NCTV, PCB301-NCTV 2.0. See Table 1 for the sequences...

Embodiment 3

[0079] Example 3: Inoculation and Identification of Infectious Clones

[0080] 1 Agrobacterium-mediated inoculation of NCTV infective clones

[0081] 1) Agrobacterium culture: pick Agrobacterium single spot or Agrobacterium glycerol in YEP liquid medium containing corresponding antibiotics, shake and culture at 28°C and 230rpm for 24-48h;

[0082] 2) Agrobacterium transfer: prepare induction medium, add MES and acetosyringone to the basic YEP medium to a final concentration of 10 mM MES (pH=5.6, mother solution concentration is 100 mM) and 20 μM acetosyringone (mother solution concentration is 200 mM);

[0083] 3) Collect the cells by centrifugation at 4,000rpm for 15min, remove the supernatant, wash with infiltration buffer (10mM MgCl 2 , 10mM MES, 200μM acetosyringone) suspension sedimentation, the bacterial suspension was placed at room temperature for starvation treatment for 3h;

[0084] 4) Select Nicotiana benthamiana plants at about 5-leaf stage, pierce the leaves, ...

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Abstract

The invention provides a construction method of infectivity clones for black nightshade curtoviruses. The method comprises the steps of respectively designing primers P1 and P2, and P3 and P4, using pLB-NCTV as formwork, and performing PCR amplification of a first copy segment of NCTV and a second cope segment of NCTV; designing primers P5 and P6, using a pCB301 carrier as a formwork, performing PCR, and using the product as a carrier framework of NCTV infectivity clones; and constructing the first copy fragment and the second copy fragment by a multi-segment homologous recombination techniqueto the carrier framework to obtain the infectivity clones containing two copy fragments of NCTV. A construction system of infectivity clones for black nightshade curtoviruses is established, so thatthe high fidelity of a virus sequence is guaranteed, the pathogenicity is high, the construction success rate of the virus infectivity clones is high, and the infection efficiency is high. Besides, the invention further provides a black nightshade curtoviruses freeze-dry positive sample, and the method has important significance in further research on the black nightshade curtoviruses.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to a method for constructing an invasive clone of Solanum nigrum virus. Background technique [0002] Solanum nigrum is a traditional Chinese medicinal material with a long history. In recent years, black nightshade has gradually attracted the attention of experts and scholars at home and abroad because of its remarkable anti-tumor effect. Recently, it was discovered that a disease of Solanum nigrum occurs widely and shows an explosive trend. The infected plants show symptoms of short plant, distorted leaves, and shrunken fruit, which seriously affects the harvest of Solanum nigrum, and the control situation is not optimistic. [0003] Solanum nigrum virus can infect Solanaceae Nightshade on a large scale under natural conditions, indicating that the virus has the risk of invading tomato, eggplant, potato, tobacco and other Solanaceae crops, but its transmission vecto...

Claims

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Application Information

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IPC IPC(8): C12N15/64C12N15/84C12N7/00C12N1/21C12Q1/70C12Q1/6806C12Q1/24C12R1/01C12R1/94
CPCC12N7/00C12N15/64C12N15/8205C12N2750/12051C12Q1/24C12Q1/6806C12Q1/701
Inventor 张蓬军孙凯杨倩倩俞晓平许益鹏郝培应
Owner CHINA JILIANG UNIV
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