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A kind of expression vector construction method and kit of citrus decay virus

A technology of decay virus and expression vector, which is applied to the construction method and kit of expression vector of citrus decay virus, and the field of virus expression vector construction, which can solve the problems of insufficient research on expression vector and the like.

Inactive Publication Date: 2015-08-19
CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the insufficient research on the existing fruit tree virus-based expression vectors in my country, the technical problem to be solved by the present invention is to provide a method for constructing an expression vector of citrus decay virus

Method used

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  • A kind of expression vector construction method and kit of citrus decay virus
  • A kind of expression vector construction method and kit of citrus decay virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Identification, isolation and preservation of citrus recession virus isolate FS-577

[0075] (1) Identification and isolation of citrus decay virus

[0076] 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, 0.02M Na 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

[0081] Embodiment 2. A method for constructing a full-length invasive clone of citrus decay virus, according to the following steps:

[0082] (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.

[0083] (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. Subsequently, 10 μL of 10× reverse transcription buffer (100 mM pH9.0 Tris-HCl, 500 mM KCl, 1% Triton X-100, 10 mM MgCl2, 0.02 M DTT), 4 U RNaseOUT (Invitrogen, USA), 2 U SuperScript was added to the reaction solution. 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 a...

Embodiment 3

[0120] Embodiment three, a kind of citrus decay virus expression vector construction method, operate according to the following steps:

[0121] (1) Identification and acquisition of citrus decay virus.

[0122] Plants infected with citrus recession virus were selected in the field according to the direct tissue point immunization method in Example 1.

[0123] (2) Total RNA extraction. Using Trizol kit (Invitrogen, USA) to extract the direct tissue point immunoassay to determine the total RNA infected with citrus recession virus strains. For the extraction method, refer to the operation manual of the Trizol kit, and details will not be repeated here.

[0124] (3) Amplification of the promoter, fragment 1 and fragment 2. Melt 8 μL of extracted total RNA at 95°C for 2 min, and mix with 190 μL of 2×one-step buffer (200 mM pH8.8 Tris-HCl, 100 mM (NH4) 2 SO 4 , 100mM KCl, 20mM MgSO 4 , 1% Triton X-100, 10mM dNTP, 0.02MDTT), 10μM forward and reverse primers (primers ZYC357 / ZYC3...

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Abstract

The invention discloses an expression vector construction method of citrus tristeza virus. The invention designs four pairs of specific primers, namely ZYC349 / ZYC350, ZYC357 / ZYC358, ZYC412 / ZYC415 and ZYC479 / ZYC480, the primers can be used for amplifying genes containing exogenous green fluorescent proteins, driving a translated citrus tristeza virus p23 gene promoter, further inserting a fragment after connection into a terminal of a p23 gene in a full-length infectious clone 35S-148 of the citrus tristeza virus by double enzyme digestion and further ensures that the citrus tristeza virus is used as an expression vector to realize stable and high-efficient expression of green fluorescent protein genes in nicotiana benthamiana for above 2 months. According to the expression vector construction method, a very important technical platform is provided for researching the distribution and the movement of the citrus tristeza virus in plants and being used as a biological reactor to realize mass expression of the high-quality proteins.

Description

technical field [0001] The invention relates to a method for constructing an expression vector of a virus, in particular to a method for constructing an expression vector of a citrus recession virus and a kit, belonging 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. A variety of virulent strains of citrus decay virus and the powerful media brown orange aphid are widely distributed in my country. With the adjustment of the citrus industry structure in the late 1980s, the proportion of pomelo and sweet orange planting increased. increased damage to oranges and some sweet oranges. [0003] With the development of molecular biology techniques, Agrobacterium expression vectors and plant virus vectors are the two main ways to express for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/65
Inventor 周彦周常勇李中安
Owner CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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