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Method for identifying tomato yellow leaf curl virus resistance

A technology for tomato yellowing leaf curl and virus, which is applied in the directions of biochemical equipment and methods, horticultural methods, and botanical equipment and methods, and can solve problems such as inability to identify TYLCV resistance.

Active Publication Date: 2012-03-28
北京中研惠农种业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the problem that traditional techniques cannot accurately identify TYLCV resistance, the inventor has carried out a large amount of research work and constructed an invasive clone (code-named TYLCV--TJ) for TYLCV in Tianjin area, which was inoculated by stem injection Tomato yellow leaf curl virus, identification of resistance to this virus in different tomato materials

Method used

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  • Method for identifying tomato yellow leaf curl virus resistance
  • Method for identifying tomato yellow leaf curl virus resistance
  • Method for identifying tomato yellow leaf curl virus resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] 1. Extraction of plant total DNAD

[0109] (1) Take the leaves 30 days after inoculation, add liquid nitrogen to grind them into powder, and quickly transfer them into 1.5 ml Eppendorf tubes;

[0110] (2) Add 800 μl of CTAB extraction buffer, mix well (CTAB is preheated in a water bath at 65°C), oscillate gently every 5 minutes, centrifuge at 12000 r / min for 20 minutes, and centrifuge for 15 minutes;

[0111] (3) Carefully draw the supernatant, add an equal volume of phenol:chloroform (400 μl each) solution, mix well, centrifuge at 12000 r / min at 4 °C for 10 min;

[0112] (4) Carefully draw the supernatant, add an equal volume of chloroform, mix well, centrifuge at 12000 r / min at 4°C for 10 min.

[0113] (5) Repeat step (4) 1-2 times until the protein layer does not appear;

[0114] (6) Take the supernatant, precipitate at -20°C for 1 hour, centrifuge at 12000 r / min at 4°C for 10 minutes;

[0115] (7) Discard the supernatant and wash the precipitate twice with 70% et...

Embodiment 2

[0167] Construction of Infectious Vector of TYLCV-TJ Isolated from Tianjin Area

[0168] The 1.4A-pMD plasmid containing 1.4A TYLCV and the plant expression vector pRI 101-An were digested with KnpI and SalI to recover the target fragment. Then use T4 ligase to connect the target fragment with the expression vector to obtain 1.4A-pRI. The ligation product was transformed into E. coli DH5α.

[0169] Digestion:

[0170] Take a 0.5 mL eppendorf tube and add the following reagents in sequence

[0171] DNA 200ng

[0172] Restriction enzyme 0.5 μL

[0173] 10× Enzyme Reaction Buffer 2 μL

[0174] Add dd HO to 20 μL

[0175] The results were checked by agarose gel electrophoresis after enzyme digestion and digestion at 37°C for 1 hour.

[0176] For connection and transformation, see 2.3

Embodiment 3

[0178] Transformation of recombinant vectors to Agrobacterium:

[0179] Preparation of Competent Cells of Agrobacterium LBA4404

[0180] 1) Agrobacterium activation: Line the Agrobacterium LBA4404 stored at -70°C on YEB solid medium containing kanamycin (50 ug / mL) and rifampicin (50 ug / mL), and place in a 28°C incubator Inverted culture in the medium for about 48h until the colony grows;

[0181] 2) Pick a single colony and inoculate it in 3 mL of YEB liquid medium containing kanamycin (50 ug / mL) and rifampicin (50 ug / mL), shake at 28°C at 220 rpm / min until OD600≈0.5;

[0182] 3) Take 1.0 mL of the bacterial solution in a 1.5 mL sterile centrifuge tube and place on ice for 10 min;

[0183] 4) Take out the centrifuge tube from ice, centrifuge at 5,000 rpm / min for 30 s in a desktop centrifuge, and discard the supernatant;

[0184] 5) Suspend the precipitate fully with 1.5 mL 0.5 mol / L NaCl, do not shake vigorously, and then place it on ice for 20 min;

[0185] 6) Centrifuge ...

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Abstract

The invention relates to a method for identifying tomato yellow leaf curl virus resistance, which enables infestive clonal construction of TYLCV in Tianjin region by aiming at a typical tomato yellow leaf curl virus sequence in Tianjin region. The resistibility of different tomato materials on the virus can be identified through inoculating tomato yellow leaf curl virus by injecting in stalks. The method avoids culturing Bemisia tabaci (Gennadius), ensures simple operation, high efficiency, high repeatability and high controllability, provides a relatively consistent inoculation pressure, obtains accurate result and is a simple and effective inoculation identification method.

Description

technical field [0001] The invention belongs to the technical field of plant protection, and relates to the research on inoculation identification of tomato yellow leaf curl virus, and more specifically relates to a rapid, simple and efficient method for identifying resistance inoculation of tomato yellow leaf curl virus in Tianjin area. Background technique [0002] Tomato yellow leaf curl virus (TYLCV) mainly harms vegetables such as tomato, pepper, eggplant, cabbage and cucumber, and cash crops such as tobacco, especially on tomato. For the occurrence and harm of TYLCV virus, how to detect the existence and occurrence of the virus accurately, quickly and efficiently is an urgent need for safe and sustainable vegetable production. [0003] At present, the methods for identifying the resistance of tomato yellow leaf curl disease include Bemisia tabaci transmission, grafting inoculation and mechanical transmission. Among them, the grafting method uses young diseased leaves ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68A01G7/06
Inventor 金凤媚刘仲齐薛俊王建江
Owner 北京中研惠农种业有限公司
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