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Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast

A green fluorescent protein, protoplast technology, applied in the field of molecular plant pathology, to shorten research time and improve work efficiency

Inactive Publication Date: 2014-06-04
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease mainly harms the base of the coconut stem, and rust-like liquid flows out from the lesion, which seriously affects the ornamental value and eventually causes the plant to die. great economic loss

Method used

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  • Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast
  • Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast
  • Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1 Detection of resistance of pathogenic bacteria to hygromycin B

[0020] Let the concentration of hygromycin B be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, pour 10 mL of CM medium (sucrose 10 g / L, acid hydrolyzed casein 6 g / L, yeast extract 6 g / L, agar powder 15 g / L), add 2μL, 4μL, 6μL, 8μL, 10μL of hygromycin B with the original concentration of 1g / 20mL , 20μL, 30μL, three times, inoculate the mycelium block in the center of the dish, seal and place it upside down in a 26°C incubator. The growth of the colony was observed every day, and the diameter of the colony was measured after 5 days. It was found that the bacteria could not grow at all on the hygromycin B plate of 40 μg / mL, and they could only grow slowly and thinly when the concentration was lower than this concentration. Therefore, the concentration of hygromycin B in the selection medium used for the transformation of S. cocois is 40-100 μg / mL.

[0021] 2 Preparation of bacte...

Embodiment 2

[0033] The difference between this implementation and Example 1 is that in step 1, the hyphae are fully washed with sterilized 0.7mol / LMgSO4.7H2O solution; 4 .7H 2 O is the osmotic agent, when using Lysing Enzymes and Drislase as lysing enzymes, when the amount of each enzyme is 7.5mg / mL, the concentration of protoplasts obtained by secondary centrifugation is 1.81×10 9 individual / mL.

Embodiment 3

[0035] The difference between this example and Example 1 is: in step 1, 1 mol / L sorbitol (Sorbitol) and 0.7 mol / L NaCl are used to fully wash the mycelia respectively; Osmotic agent, when using 10mg / mL Lysing Enzymes and Drislase as lysing enzymes, the concentration of protoplasts obtained by secondary centrifugation is 6.15×10 8 pcs / mL; if Sorbitol (Sorbitol) is used as the osmotic agent and Lysing Enzymes 10 mg / mL (Lysing Enzymes) is used as the lysing enzyme in step 2, 1.73×10 9 protoplasts / mL. In summary, the present invention determines the suitable conditions for the formation of protoplasts, and the stabilizer and enzyme can be selected. Based on the principles of timeliness and economy, it is best to choose 1mol / L mannitol as the osmotic agent and 7.5mg / mL disintegrating enzyme as the lyase. It shows that the yield of protoplast is the highest and the time is the shortest. In terms of protoplast regeneration rate, there is little difference among the above three imp...

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Abstract

The invention belongs to the field of molecular plant pathology, and particularly relates to a preparation method of a green fluorescent protein marked coconut stem bleeding disease bacterium protoplast. The preparation method has the beneficial effects that appropriate conditions for protoplast formation are determined, the high-quality protoplast is prepared by using 1mol / L mannitol as an osmotic stabilizer and 7.5mg / mL driselase as lyase, GFP (green fluorescent protein) is transferred to a coconut stem bleeding disease bacterium so as to obtain a stable genetic GFP pathogenic bacterium strain; therefore, a solid foundation is laid for genetic operations of the disease bacterium, such as GFP transformation and genetic expression, the cost is saved, the working efficiency can be greatly improved, and the research time is shortened; and meanwhile, the green fluorescent protein marked coconut stem bleeding disease bacterium facilitate the research on histopathology, disease epidemiology and the like of the disease.

Description

technical field [0001] The invention belongs to the field of molecular phytopathology, and in particular relates to a method for preparing protoplasts of Coconut stem-diarrhea bacteria marked by green fluorescent protein. Background technique [0002] Coconut stem bleeding disease is becoming more and more serious in my country, especially in Hainan, and the incidence rate in Qionghai, Wanning, and Tunchang is increasing year by year. The disease mainly harms the base of the coconut stem, and rust-like liquid flows out from the lesion, which seriously affects the ornamental value and eventually causes the plant to die. great economic loss. Its causative agent is A. mirabilis ( Ceratocystis paradoxa ) of the asexual generation of Moniligo mirabilis ( Thielaviopsis paradoxa ). [0003] Green Fluorescent Protein (GFP for short) is a protein discovered in jellyfish abundant in the west coast of the United States in 1962. It is not only non-toxic, but also can emit light b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N15/80C12R1/645
Inventor 鲁国东牛晓庆王宗华余凤玉李亚郑华伟朱辉宋薇薇唐庆华
Owner FUJIAN AGRI & FORESTRY UNIV
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