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Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof

A technology of geminiviruses and plants, applied in the biological field, can solve problems affecting the accuracy of experimental results, cumbersome steps, and reducing experimental efficiency, etc., and achieve the effects of large-scale promotion, accurate results, and good repeatability

Inactive Publication Date: 2013-11-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a long time, the research on geminivirus has mostly used the method of bemisia tabaci transmission and inoculation, but this method requires bemisia tabaci to complete the virus transmission, so there are many problems that are difficult to overcome: 1. Bemisia tabaci are difficult to raise; 2. Whether the whitefly is carrying the virus, whether the virus is pure and single, and the detection of the virus-carrying rate of the whitefly is difficult, etc., resulting in that the source of the virus in each test cannot be unified; 4. The transmission efficiency of Bemisia tabaci is even more difficult to control, which affects the accuracy of the experimental results; 5. Since Bemisia tabaci can transmit a variety of geminiviruses and other diseases at the same time, the inoculation results are often very disturbing
The recently developed Agrobacterium liquid injection method needs to use the inoculation buffer for post-processing the bacterial liquid, the steps are cumbersome, the dependence on the instrument is strong, the operation is inconvenient, and the experimental efficiency is reduced.
In addition, the syringes and excess bacterial fluid consumed during the experiment caused environmental pollution, which is not conducive to environmental protection, and the injection inoculation method is poorly operable for plants with thick stems, which affects the efficiency of geminivirus inoculation

Method used

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  • Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof
  • Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof
  • Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Acquisition of Geminivirus Infectious Clone Inoculation Virus Source

[0042] Collect geminivirus samples from the field and extract total genomic DNA; design geminivirus-specific primers, PCR amplify the full genome sequence of the virus, and clone the amplified product into T-Vector; perform PCR screening and sequence determination on the cloned product; use restrictions The 1.2-2 repeated sequences of the full length of the viral genome were constructed on the improved plant expression vector pBinplus by the method of restriction enzyme digestion, and the infectious clones with geminiviruses were obtained; It was introduced into the Agrobacterium cell EHA105 by electric shock to obtain the source of inoculated virus.

[0043] The above method of inoculating the virus source can refer to the thesis published by the inventor of the patent and the graduation thesis of the graduate student supervised (Chinese papaya leaf curl virus source (for specific methods...

Embodiment 2

[0044] Example 2: Pathogenicity determination of geminivirus and research on virus gene function (taking Taiwan tomato leaf curl virus as an example)

[0045] The Taiwan tomato leaf curl virus (Tomato leaf curl Taiwan virus, ToLCTWV) invasive clone Agrobacterium was inoculated into YEP liquid medium containing 100 mg / l kanamycin and 50 mg / l rifampicin for activation, 28°C 200rpm Grow to OD 600 Reach 0.8-1.0. Take 50ul of the above-mentioned activated bacterial solution into the YEP solid medium containing 100mg / l kanamycin and 50mg / l rifampicin, spread evenly on the plate, and culture at 28°C for 24-48h, until the colony covers the whole flat. Pick plate colonies, and inoculate the original host tomato, and other hosts Nicotiana benthamiana, Petunia, and Nicotiana cordata by pricking with a toothpick. ToLCTWV showed strong pathogenicity on its original host tomato, and all inoculated plants showed symptoms. 14 days after inoculation, the tomato plants exhibited leaf roll-d...

Embodiment 3

[0047] Embodiment 3: the determination of the resistance variety of plant anti-geminivirus

[0048] Taking tomato varieties Zhe A, Zhe B, Zhe C and Ruby as the research objects, the phloem inoculation of plate colony toothpick was pricked to identify the resistance of each tomato variety to Chinese papaya leaf curl virus, tomato yellow leaf curl virus, and Chinese tomato yellow leaf curl virus. Leaf Curl Virus, Thai Tomato Leaf Curl Virus, and Taiwan Tomato Leaf Curl Virus.

[0049] 1. Experimental treatment

[0050] a) Inoculate Agrobacterium EHA105 containing invasive clones of Chinese papaya leaf curl virus, tomato yellow leaf curl virus, Chinese tomato yellow leaf curl virus, Thai tomato yellow leaf curl virus, and Taiwan tomato leaf curl virus containing 100mg / 1 kanamycin and 50mg / l rifampicin YEP liquid medium for activation, 28 ° C 200rpm culture to OD 600 Reach 0.8-1.0.

[0051] Take 50ul of the above-mentioned activated bacterial solution into the YEP solid me...

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Abstract

The invention discloses a method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and an application of the method. The method comprises the steps of coating geminivirus infectious clone-containing Agrobacterium tumefaciens EHA105 on YEP solid culture medium containing 100 mg / L kanamycin and 50 mg / L rifampicin; culturing into colony at 28 DEG C; picking colony with toothpicks; piercing 3-4 points on phloem of plant after three-leaf stage, at position 1-2 cm from root; culturing inoculated plant in a greenhouse; and observing virus symptom. The inoculation method provided by the invention has the advantages of no need for raising Bemisia tabaci, high inoculation efficiency and repeatability, simple and rapid operation, and suitability for large-scale popularization. The method provided by the invention is applicable to 16 kinds of geminivirus with severe breakout in our country. The inoculated plants are in families of Solanaceae, Cucurbitaceae and Convolvulaceae. The method can be used for pathogenicity identification of geminivirus and virus gene function research, anti-geminivirus plant variety determination and its breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for pricking and inoculating plants with a solid bacterium colony containing geminivirus infective clones and an application thereof. Background technique [0002] Geminiviruses are a class of plant viruses that occur widely. With global warming, changes in agricultural farming systems, the rapid strengthening of international trade activities and the unprecedented expansion of insect vectors around the world, geminiviruses are widely occurring around the world. At present, geminivirus has been widely distributed and prevalent in Asia, central America, central and northern South Africa, and the Mediterranean, and is further expanding to surrounding uninfected areas. According to preliminary statistics, cotton, cassava, tomato and other crops in at least 40 countries have suffered devastating damage from such viruses. [0003] Since the 1990s, with the global warming and the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34
Inventor 周雪平谢艳矫晓阳
Owner ZHEJIANG UNIV
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