A kind of preparation method of anti-tobacco potato virus y vaccine

A virus vaccine and potato technology, applied in botany equipment and methods, disinfectants, chemicals for biological control, etc., can solve the problems of PVY virus control, environmental biological safety hazards, pesticide residues, etc., and achieve simple and fast operation , low cost, reduced duplication effect

Inactive Publication Date: 2018-12-21
中国烟草总公司黑龙江省公司烟草科学研究所 +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Tobacco PVY virus has no effective prevention and control agents at present, and the production is mainly based on prevention. Some conventional antivirus agents are affected by factors such as the environment. On the one hand, it is difficult to effectively prevent and control PVY virus. Residues, which are very harmful to the environment and other biological safety

Method used

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  • A kind of preparation method of anti-tobacco potato virus y vaccine
  • A kind of preparation method of anti-tobacco potato virus y vaccine
  • A kind of preparation method of anti-tobacco potato virus y vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Preparation of c-DNA template

[0037] RNeasy Plant Mini kit from QIAGEN was used to extract RNA from tobacco LJ925 fresh leaves, and c-DNA was synthesized by reverse transcription using TaKaRa RNA PCR Kit Ver.3.0 kit as a template. For specific operations, refer to the instructions of the kit.

[0038] 2. Amplification of eIF4E-6 gene

[0039] Add 2.5 μL of 10×PCR buffer (Buffer), 0.5 μL of 10 pmol / μL of PVX-BF and PVX-MR primers, 0.5 μL of pfu DNA polymerase of 2.5 U / μL, dNTPs (per Seed dNTP 10mM) 1μL, c-DNA template 2μL, ultrapure water 18μL. The PCR amplification program is shown in Table 1:

[0040] Table 1: PCR reaction conditions

[0041]

[0042] 3. Construction of pMD18-T-eIF4E-6 plasmid

[0043] The amplified product of 25 μL eIF4E-6 gene was separated by 1.5% agarose electrophoresis, and the target fragment was recovered by gel cutting in an ultraviolet analyzer; the target fragment was recovered and purified and cloned into pMD18-T Simple Vector (...

Embodiment 2

[0048] Example 2: Amplification of Vaccines Against Tobacco Potato Virus Y

[0049] Take 50 μL of the anti-tobacco potato virus Y vaccine prepared in Example 1, respectively, and add it to 50 mL of YEP liquid medium (containing 50 mg / mL of kanamycin, take 10 mL after each bacterial solution is shaken), shake the bacteria at 28°C until logarithmic growth During the period, after centrifugation at 8000rpm for 5 minutes, the cells were osmotic medium buffer (10mmol / L MES, 10mmol / L MgCl 2 , 100 μmol / L acetosyringone) was suspended, the concentration of the bacterial solution was adjusted to OD600 = 1.5, and stood at 28°C for 3 hours for use.

Embodiment 3

[0050] Embodiment 3: the inoculation method of tobacco potato virus Y and the use of anti-tobacco potato virus Y vaccine

[0051] The anti-tobacco potato virus Y vaccine suspension in embodiment 3 is diluted 50 times, before tobacco seedlings are transplanted, spray tobacco kind LJ911 (sensitivity to PVY) seedling (5 true leaf stage) twice, spray interval 5 days, as test Group. At the same time, no inoculation and inoculation of PVX empty vector controls were set. Nine pots were planted in each treatment, and the PVY virus was inoculated by rubbing when the tobacco seedlings grew to the group tree stage (12 true leaf stage). The inoculation method is: take the fresh PVY diseased leaves bred by LJ912 (anti-TMV) tobacco seedlings, put them into a mortar and mash them, add phosphate buffer solution equal to the weight of the diseased leaves twice as much (0.5 g of sodium sulfite, 5 g of potassium dihydrogen phosphate , dissolved in 500ml of water) ground and smashed into a past...

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Abstract

The invention discloses a preparation method of an anti-tobacco potato Y virus vaccine. The method includes the following steps that 1, pCaPVX440-eIF4E-6 plasmids are prepared; 2, the pCaPVX440-eIF4E-6 is transferred to agrobacterium to prepare the anti-tobacco potato Y virus vaccine. The anti-tobacco potato Y virus vaccine prepared by means of the preparation method is suitable for prevention and control of tobacco PVY viruses and has an important practical value for prevention and control of diseases caused by the PVY viruses in the tobacco leaf production process. Compared with the prior art, the virus vaccine is good in specificity, good in safety, simple, convenient and quick to operate and low in cost.

Description

technical field [0001] The invention relates to the preparation and application of a tobacco antiviral vaccine, belonging to the field of plant disease prevention and protection. Background technique [0002] Potato Virus Y (PVY) is a typical representative of Potatovirus Y (Potyviruses), which was first discovered on potatoes by K.M.Smith in 1931. Potato virus Y is mainly transmitted by aphids under natural conditions, and can infect Solanaceae plants worldwide. It is one of the important viral diseases that commonly occur in tobacco production in my country. After tobacco is infected with Potato Y virus, it shows obvious yellowing, dwarfing, vein necrosis, mottled leaves, withering of the whole plant, serious browning and necrosis of the root system, and the leaves lose their baking value, resulting in serious reduction in yield and quality. until extinction. [0003] The use of virus resistance genes for virus control is an important research method for crop disease res...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74A01N63/00A01P1/00
Inventor 乔婵李若万秀清李向东李丽杰孙健桐郭思达王春军元野贺国强颜培强郭振楠邱恩建李尊强孙宏伟焦玉生李恒全刘德育陈荣平黄磊张海霞魏继承
Owner 中国烟草总公司黑龙江省公司烟草科学研究所
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