Agrobacterium tumefacien mediated genetic transformation method for ustilago esculenta

A genetic transformation method, the technology of Agrobacterium tumefaciens, applied in the directions of microorganism-based methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc.

Inactive Publication Date: 2017-10-27
广西壮族自治区农业科学院生物技术研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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So far, there is no report on the genetic tr...

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  • Agrobacterium tumefacien mediated genetic transformation method for ustilago esculenta
  • Agrobacterium tumefacien mediated genetic transformation method for ustilago esculenta
  • Agrobacterium tumefacien mediated genetic transformation method for ustilago esculenta

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Embodiment 1

[0024] Example 1 Obtainment of T-DNA Insertion Transformants of Zizania smut

[0025] Preparation of Zizania spores

[0026] After cultivating the Zizania smut fungus on YePSA solid medium (yeast extraction 1%, peptide 2%, sugar 2%, agar 2%) for 2 days, inoculate the Zizania smut fungus into YePS liquid medium (yeast extraction 1%, peptone 2 %, sugar 2%), shaker 200rpm, culture for 2 days, then centrifuge at 5000rpm to collect the bacteria, add ddH 2 O 2 Make 1×10 7 Pcs / mL spore suspension ( figure 1 )), to obtain a spore suspension of Zizania spp. for use. All the above operations are carried out under sterile conditions.

[0027] Preparation of carrier Agrobacterium

[0028] Using plasmids pEX2-eGFP and pEX2-RFP as transformation vectors, the pEX2-eGFP vector carries the green fluorescent protein gene eGFP and the hygromycin resistance screening gene hph( figure 2 A), pEX2-RFP vector carries red fluorescent protein gene RFP and hygromycin resistance screening gene hph( figure 2...

Embodiment 2

[0032] Example 2 Identification of Zizania smutella transformants

[0033] 1) Fluorescence microscope identification

[0034] Place the conidia of Zizania smutella transformants under a fluorescence microscope to observe the fluorescence. No fluorescence signal was detected in the wild-type Zizania spp., the transformant transformed by Agrobacterium emits green or red light under the excitation light of the fluorescence microscope, which proves that the green fluorescent protein GFP and red fluorescent protein RFP of Agrobacterium have been successfully transferred. And expressed in Zizania smut ( image 3 ).

[0035] 2) PCR verification of Zizania smutella transformants

[0036] Design primer hph-F: ATGAAAAAGCCTGAACTCACCGC (SEQ.ID.No.2) / hph- using the hygromycin gene (hph(SEQ.ID.No.1)) on the binary vector pEX2-eGFP and pEX2-RFP as a template. R: GCTGCTCCATACAAGCCAACCAC (SEQ. ID. No. 3) PCR amplification was performed on the transformant (the length of the amplified product was abo...

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Abstract

The invention discloses an agrobacterium tumefacien mediated genetic transformation method for ustilago esculenta, wherein an ustilago esculenta spore is taken as a transformation acceptor material, the agrobacterium tumefacien containing a binary vector and the ustilago esculenta spore are mixed, co-cultured, and screened by an antibiotic, so as to obtain a resistant transformant. The method is simple in operation, high in transformation efficiency and high in stability, an ustilago esculenta mutant library is created according to the method, the rick screening bacteria source is provided for screening a binary transformation mutant of the ustilago esculenta, meanwhile, a means is provided for cloning a binary transformation gene of the ustilago esculenta and an interaction related gene of the ustilago esculenta and a zizania aquatica plant, and the method is of great significances for clarifying the stem swelling mechanism of the zizania aquatica and promoting the breeding of the zizania aquatica.

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 Zizania smut fungus mediated by Agrobacterium tumefaciens. Background technique [0002] Zizania latifolia (Zizania latifolia), also known as wild rice, is a perennial root herb of the genus Zizania in the Gramineae family. Its meat is fresh and tender, rich in nutrients and various medicinal ingredients. It is widely cultivated in the Yangtze River basin and areas to the south, and is known in Liuzhou, Guilin, Hezhou and other regions in Guangxi. With the continuous development of the water bamboo industry and the continuous expansion of the planting area, the water bamboo has become the second largest aquatic vegetable in my country. [0003] The Ustilago esculenta belongs to the Basidiomycota Family Ustilago. The study found that Ustilago esculenta is an endophytic fungus of the Zizania plant. The ability of th...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/645
CPCC12N15/80
Inventor 颜梅新江文欧昆鹏桂杰董伟清高美萍何芳练张尚文王艳
Owner 广西壮族自治区农业科学院生物技术研究所
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