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Construction method and kit of full-length infectious clone of citrus tristeza virus

A technique for decaying viruses, constructing methods, applied in the field of biology

Inactive Publication Date: 2014-03-19
CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Such studies have not yet been carried out in our country

Method used

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  • Construction method and kit of full-length infectious clone of citrus tristeza virus
  • Construction method and kit of full-length infectious clone of citrus tristeza virus
  • Construction method and kit of full-length infectious clone of citrus tristeza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Identification, isolation and preservation of citrus recession virus isolate FS-577

[0073] (1) Identification and isolation of citrus decay virus

[0074] Mature shoots with leaves were collected from four different directions of the plants in the field. The method of direct tissue spot immunization is used for detection, and the specific operation is as follows: use a razor blade to cross-cut the stem or petiole of the sample, and then press it evenly on the nitrocellulose membrane, set positive and negative controls, and dry at room temperature for more than 15 minutes . Place the blotted membrane in 0.02M pH7.2 phosphate buffer containing 1%BSA (0.13M NaCl, 0.02MNa 2 HPO 4 12H 2 O, 14mM KH 2 PO 4 , 20mM KCl, 20mM NaN 3 ) was directly added to 1000:1 (v / v) alkaline phosphatase (Ap)-labeled CTV-specific antibody IgG (BioRad, USA) after blocking for 1 h, and the reaction was shaken at room temperature for 2 h. Pour off the buffer containing Ap-IgG, ...

Embodiment 2

[0078] Embodiment 2. A method for constructing a full-length invasive clone of citrus decay virus, according to the following steps:

[0079] (1) Total RNA extraction: Trizol kit (Invitrogen, USA) was used to extract the total RNA of citrus recession virus isolate FS-577 in the diseased plants. For the extraction method, refer to the operation manual of the Trizol kit, and details will not be repeated here.

[0080] (2) Synthesis of first-strand cDNA: 8 μL of extracted total RNA was mixed with 1 μL of 50 ng / μL random primer (Promega, USA), 1 μL of 10 mM dNTP, reacted at 65°C for 5 min, and placed on ice for 1 min. Then add 10 μL of 10× reverse transcription buffer (100 mM pH9.0 Tris-HCl, 500 mM KCl, 1% Triton X-100, 10 mM MgCl 2 , 0.02M DTT), 4U RNaseOUT (Invitrogen, USA), 2U SuperScript III RT (Invitrogen, USA). After mixing, react at 25°C for 10 minutes, at 50°C for 50 minutes, and at 85°C for 5 minutes. Then place it on ice for at least 1 min, then add 1 μL 2U / μL RNaseH ...

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Abstract

The invention discloses a construction method and a kit of a full-length infectious clone of citrus tristeza virus and belongs to the technical field of biology. The invention designs six pairs of specific primers against the complete sequence of a citrus tristeza virus separated strain FS-577, and the amplification region of the primers covers the complete sequence of the FS-577. Compared with a wild type FS-577, the full-length infectious clone obtained after an amplification fragment is subjected to double enzyme digestion and then sequentially inserted into a binary vector pUC119-delta 9 after transformation has completely consistent sequence, infection ability, host range and other characters. The kit established by utilizing the method comprises first chain cDNA (complementary deoxyribonucleic acid) synthesis, PCR (polymerase chain reaction) amplification and double enzyme digestion buffer solutions, a link reaction buffer solution and the like, and the kit can be used for constructing the full-length infectious clone of the citrus tristeza virus. According to the construction method, a very important technical platform is provided for researching pathopoiesis, aphid transmission and other mechanisms of the citrus tristeza virus and controlling citrus tristeza disease.

Description

technical field [0001] The invention relates to a method and a kit for constructing a full-length infectious clone of a virus, in particular to a method for constructing a full-length infectious clone of a citrus decay virus and a kit, and belongs to the field of biotechnology. Background technique [0002] China's citrus planting area and output both rank first in the world. Citrus tristeza virus (CTV) caused by Citrus tristeza virus (CTV) is an important worldwide citrus disease, which is transmitted by aphids and diseased seedlings. Various virulent strains of citrus decay virus and brown aphid, which are powerful mediators, are widely distributed in my country. With the adjustment of the citrus industry structure in my country in the late 1980s, the proportion of pomelo and sweet orange planting increased, and citrus declined. Pomelo and some sweet oranges are increasingly infested by the disease. [0003] Infectious vectors of plant viruses are a crucial platform for t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66
Inventor 周彦周常勇李中安
Owner CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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