Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum

A genetic transformation method, the technology of Agrobacterium tumefaciens, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as the difficulty of genetic transformation of Sclerotinia sclerotiorum, and achieve simple preparation technology , easy operation and high conversion efficiency

Inactive Publication Date: 2010-10-20
HUAZHONG AGRI UNIV
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Problems solved by technology

The present invention uses the protoplast of Sclerotinia sclerotiorum as the acceptor and Agrobacterium tumefaciens as the mediator, and overcomes the difficulty in the genetic transformation of Sclerotinia sclerotiorum caused by the fact that the mycelial cells of Sclerotinia sclerotiorum are multinucleated and the ascospores are difficult to obtain in large quantities. Problem, provide a kind of simple and convenient, the genetic transformation method of sclerotinia with stable transformation efficiency

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  • Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum
  • Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum
  • Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum

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Embodiment 1

[0070] Embodiment 1: the construction of the T-DNA random insertion mutant library of Sclerotinia bacterial strain Sunf-M

[0071] 1. Preparation of protoplasts from the transformed recipient S. sclerotiorum strain Sunf-M

[0072] Inoculate the S. sclerotiorum strain Sunf-M (Xie Jun et al., Journal of General Virology. 2006, 87, 241-249) on the PDA medium, culture it at 20°C, and continuously pick the vigorously growing S. sclerotiorum Sunf -M colony edge hyphae blocks were transferred to the 2nd generation activated strain.

[0073] Take 3 mycelial pieces at the edge of the activated S. sclerotiorum strain Sunf-M colony and inoculate them on a PDA plate covered with cellophane. After 48 hours, the mycelium was collected, ground with a sterilized mortar, added 3ml of PDB, and mixed with a pipette equipped with a sterilized tip. Aspirate mycelial fragments and inoculate them into 100ml of PDB medium, shake at 150r / min at 20°C for 24h, then centrifuge at 6000r / min for 10min, a...

Embodiment 2

[0111] Embodiment 2: Feasibility analysis embodiment

[0112] Although S. sclerotiorum protoplasts are easier to transform than hyphae, and easier to obtain than ascospores, the preparation of protoplasts for S. sclerotiorum transformation has been reported to prepare a variety of different solutions, and it needs to be washed several times with different solutions. Mycelia or protoplasts (Rollins, 2003; Yang Lei et al., 2006), so the complicated preparation of S. sclerotiorum protoplasts has become the biggest disadvantage of using protoplasts as transformation recipients. The invention optimizes conditions such as protoplast preparation and protoplast preservation. The preparation method of S. sclerotiorum protoplast provided in the invention only needs to prepare MgSO 4 and STC solution, and only need to use MgSO 4 The mycelium was washed once with liquid, and the obtained protoplasts did not need to be washed and other treatments, and the protoplasts could be preserved i...

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Abstract

The invention provides a method for the genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum. The method comprises the following steps: mixing and co-culturing the agrobacterium tumefaciens containing binary vectors and the protoplasts of the sclerotinia sclerotiorum; and screening by using antibiotics to obtain transformants, the transformation efficiency of the method reaches 1/5.9*104 protoplasts. By using the protoplasts of the sclerotinia sclerotiorum as the receptor and using the agrobacterium tumefaciens as the mediator, the method of the invention overcomes the problem of the difficult genetic transformation of sclerotinia sclerotiorum, which is resulted from the multinucleate hypha cells and the difficulty in obtaining a large amount of ascospore, and has the advantages of simple method and stable transformation efficiency. Meanwhile, the invention further provides a simple and effective method for preparing the protoplasts of sclerotinia sclerotiorum.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a method for genetic transformation of S. sclerotiorum mediated by Agrobacterium tumefaciens. Background technique [0002] Sclerotinia sclerotiorum (Lib.) de Bary) is an important plant pathogenic fungus distributed worldwide, with a wide range of hosts, and can parasitize more than 400 species of plants in 75 families (Boland and Hall, 1994). It mainly parasitizes dicotyledonous plants such as beans and cruciferous plants such as rapeseed, and can also parasitize some monocotyledonous plants. It causes sclerotinia in important oil crops and vegetable crops such as rape and beans in my country. The incidence of Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum in the Yangtze River Basin of my country is about 30% in normal years, and more than 80% in severe cases, and the yield is reduced by 20%-70%. Due to the lack of disease-resist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12R1/645
Inventor 姜道宏肖雪琼谢甲涛付艳苹程家森
Owner HUAZHONG AGRI UNIV
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