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Agrobacterium tumefaciens-mediated transformation method for Tilletia contraversa Kuhu

A technology mediated by Agrobacterium dwarfus and Agrobacterium tumefaciens is applied in the direction of introducing foreign genetic material using a vector, recombinant DNA technology, etc., and can solve problems such as the lack of establishment of a genetic transformation system.

Active Publication Date: 2018-12-07
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0004] At present, most of the research on T. tritici is focused on morphological identification and classification, and the genetic transformation system of Agrobacterium tumefaciens mediated by T. tritici has not yet been established.

Method used

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  • Agrobacterium tumefaciens-mediated transformation method for Tilletia contraversa Kuhu
  • Agrobacterium tumefaciens-mediated transformation method for Tilletia contraversa Kuhu
  • Agrobacterium tumefaciens-mediated transformation method for Tilletia contraversa Kuhu

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Embodiment 1

[0043] Embodiment 1, establishment of Agrobacterium-mediated transformation system of Tilletia dwarfis

[0044] 1. Germination of Teliospores of Ustilago dwarfis

[0045] Cultivate and germinate teliospores in the soil extract medium, the steps are as follows:

[0046] Prepare teliospore suspension at a concentration of 1×10 6 Individuals / mL, take 220 μL teliospore suspension and spread it on the soil extract medium, place it in an artificial climate growth incubator with a relative humidity of 50% at 5°C and carry out full-light cultivation (24h light a day, light intensity 1Lx) After 30 days, the germination of TCK teliospores was observed with a fully automatic research-grade inverted microscope. The genetic transformation was carried out when the number of infected hyphae reached the peak of germination (germination rate ≥ 60%).

[0047] 2. Prepare the mycelia liquid of Tilletia tritici

[0048] Preparation of TCK mycelium liquid: wash the grown mycelium with sterile w...

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Abstract

The invention relates to genetic transformation of Tilletia contraversa Kuhu, and specially relates to an agrobacterium tumefaciens-mediated transformation method for the Tilletia contraversa Kuhu. The method includes the following steps: culturing TCK teliospores, collecting mycelia when the germination rate is more than or equal to 60%, and preparing a mycelium liquid with 1*10<6> / ml; inoculating the agrobacterium tumefaciens in an LB culture medium, performing shake cultivation at 28 DEG C for 2-3 d, diluting with an IM culture medium until OD<600> is 0.15-0.35, continuing to culture for 7-8 h, and diluting with the IM medium to the OD<600> is 0.53-0.59; laying a layer of sterile cellophane on a CM medium, isovolumetrically mixing an agrobacterium tumefaciens liquid with the mycelium liquid, coating the cellophane with the mixture, and performing darkness culture at 20-22 DEG C for 48 h; and then transferring the cellophane to a new CM medium to make one side coated with a bacterialsolution attach to the culture medium, and performing the darkness culture at 5 DEG C until a bacterium colony grows. According to the method, foreign genes can be successfully transferred into a genome of the Tilletia contraversa Kuhu and can be stably inherited.

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 controversa Kühn (TCK), caused by Tilletia controversa Kühn (TCK), is one of the most destructive wheat diseases in the world. Trimethylamine can reduce the yield and quality of wheat, causing devastating harm to wheat production. Therefore, the control of wheat dwarf smut is urgent. The pathogenic gene and pathogenic molecular mechanism of T. tritici are still unclear, but the construction of a large-capacity T. dwarf transformant library is the key to study the functional genes of the pathogen and the interaction between wheat and T. dwarf. Foundation. [0003] Among many fungal genetic transformation methods, Agrobacterium tumefaciens-mediated transformation (ATMT) not only has the advantages of simple operation, high transformation efficiency, and un...

Claims

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Application Information

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IPC IPC(8): C12N15/80
CPCC12N15/80
Inventor 高利陈万权刘俭俭秦丹丹陈德来周磊邵文达刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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