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Agrobacterium-mediated tilletia foetida transformation method

A technology mediated by Agrobacterium and Agrobacterium, which can be applied to microorganism-based methods, biochemical equipment and methods, fungi, etc., and can solve problems such as difficulties in genetic transformation of wheat smut.

Active Publication Date: 2018-11-16
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genetic transformation of Tilletia tritici mediated by Agrobacterium tumefaciens is very difficult, and there are no related reports on the genetic transformation system of Agrobacterium tumefaciens mediated by Tilletia tritici

Method used

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  • Agrobacterium-mediated tilletia foetida transformation method
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  • Agrobacterium-mediated tilletia foetida transformation method

Examples

Experimental program
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Effect test

preparation example Construction

[0031] The preparation method of 2% water agar medium: weigh 20g of agar powder, add water to make up to 1L, and sterilize by high pressure steam at 121°C.

[0032] IM medium (Induction Medium, induction medium): K-buffer (pH 4.9) 0.8mL, M-Nbuffer 20mL, 1% CaCl 2 2H 2 O (w / v) 1 mL, 20% glucose (w / v) 10 mL, 20% NH 4 NO 3 (w / v) 2.5mL, 50% glycerin (v / v) 10mL, supplemented with distilled water to 1000mL, and then sterilized by high-pressure steam at 121°C for 30min after dispensing. Before using the medium, add 4 μL 0.2mol / L AS (acetosyringone) per mL, 1 μL 0.01% FeSO 4 (w / v), 10 μL of 100 mg / mL 2-(N-morpholine)ethanesulfonic acid (MES). Wherein, w / v all represent g / ml.

[0033] K-buffer formula: with ddH 2 O is a solvent, containing 200g / L K 2 HPO 4 , 145g / L KH 2 PO 4 .

[0034] M-N buffer formula: ddH 2 O is the solvent, containing 30g / L MgSO 4 ·7H 2 O, 15g / L NaCl.

[0035] CM medium (Complete Medium, complete medium): yeast extract 1g, enzyme hydrolyzed casein 0...

Embodiment 1

[0042] Example 1. Genetic Transformation of Agrobacterium-mediated Tilletia tritici

[0043] 1. Germination of Teliospores of Tilletia tritici

[0044] Cultivate germinating teliospores in 2% water agar medium, the steps are as follows:

[0045] Prepare teliospore suspension, the concentration is (100-105) × 10 4 Individual / mL, take 220 μ L teliospore suspension and spread on the water agar medium, place 16 ℃, relative humidity is 80% artificial climate growth incubator and culture in full light (24h a day light, light intensity 1Lx), 3 Two days later, the germination was observed under a full-automatic research-grade inverted microscope, and the teliospores could be collected for genetic transformation when they reached the peak of germination (germination rate ≥ 60%).

[0046] 2. Preparation of mycelia liquid of Tilletia tritici

[0047] Preparation of mycelia liquid: wash the grown mycelium with sterile water under the ultra-clean workbench with a sterile triangular rod,...

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Abstract

The invention relates to tilletia foetida genetic transformation, and more specifically relates to an agrobacterium-mediated tilletia foetida transformation method. The method comprises the followingsteps: culturing TFL teliospore, when a germination rate is greater than or equal to 60%, collecting mycelia, and preparing 1*106 / ml mycelia liquid; inoculating agrobacterium in a LB medium, performing oscillation culture for 2-3 d at the temperature of 28 DEG C, diluting the material to OD600 being 0.5 by using an IM medium, continuously culturing for 7-8 h, and using the IM medium for diluting the material to OD600 being 0.45-0.5; spreading a layer of aseptic glassine paper at the CM medium, performing isopyknic mixing of agrobacterium bacteria liquid and mycelia liquid and adding AS to 200[mu]mol / l, coating the material at the glassine paper, culturing the material in dark for 24 h at the temperature of 22 DEG C; transferring the glassine paper to a novel CM medium, applying one surface coated with the bacteria liquid to the medium, and culturing the material at the temperature of 16 DEG C until bacterium colony is grown. The method successfully transfers the exogenous gene to thetilletia foetida genome and realizes stable heredity.

Description

technical field [0001] The invention relates to the genetic transformation of Tilletia tritici, in particular to an Agrobacterium-mediated transformation method for Tilletia tritici. Background technique [0002] Tilletia foetida (Wallr.) Liro, TFL) caused by Tilletia foetida (Wallr.) Liro, is one of the most destructive wheat diseases in the world. Supplementary agricultural phytosanitary pests. Tilletia glabris releases trimethylamine with a fishy smell through the production of toxic black fungus teliospores, causing reduced yield and quality of wheat, causing devastating damage to wheat production. If the spore content of this pathogen exceeds 0.6%, severe poisoning can be caused, and severe poisoning symptoms such as nausea, vomiting and even coma can be caused by humans and animals after eating (Goats, 1999; Hoffman, 1982). Therefore, the control of wheat light smut is urgent. [0003] The pathogenic gene and pathogenic molecular mechanism of Tilletia tritici are st...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/645
CPCC12N15/80
Inventor 高利陈万权秦丹丹陈德来魏晓庆刘俭俭刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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