Agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method

Abstract

The invention discloses an agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method and belongs to the field of genetic transformation of tuber melanosporum. The agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method disclosed by the invention comprises the following steps: (1) preparing a tuber melanosporum spore suspension; (2) transforming a binary vector plasmid carrying a target gene to agrobacterium tumefaciens to obtain a positive transformant of agrobacterium tumefaciens so as to prepare a bacterial liquid; (3) mixing the bacterial liquid prepared in the step (2) and the tuber melanosporum spore suspension prepared in the step (1) and co-culturing; and (4) selectively culturing to screen the transformant of agrobacterium tumefaciens and identifying to obtain. By taking spores of tuber melanosporum as a receptor and agrobacterium tumefaciens as a mediator, the method disclosed by the invention has the advantages of being simple and convenient, high and stable in transformation efficiency, high in frequency that the transformant is monoclonal and the like and is suitable for functional genomical and biological studies of tuber melanosporum and the like.

Description

technical field

[0001] The invention relates to a method for transforming black truffle, in particular to a method for genetic transformation of black truffle mediated by Agrobacterium tumefaciens, and belongs to the field of genetic transformation of black truffle. Background technique

[0002] Black spore truffle (Tuber melanosporum) is an ectomycorrhizal edible fungus native to France and symbiotic with the roots of oak, oak and other trees. It is famous for its unique fragrance and "diamond" status attention. The completion of the genome sequencing of Niger truffle has promoted researchers' understanding of the biology of Niger truffle at the molecular level, but because the niger truffle has not yet established a mature genetic operating system, it greatly limits the functional genomics of Niger truffle. Research.

[0003] The Agrobacterium tumefaciens-mediated filamentous fungal transformation method developed in recent years has the following three advantages: First...

Examples

Embodiment 1

[0042] The construction of embodiment 1 black spore truffle mutant library

[0043] 1. Experimental method

[0044] 1.1 Preparation of spores of Truffle nigrosporum

[0045] (1) Inoculate the mycelia of Trichomonas nigeri on PDA medium, and culture and activate at 28°C for 3 days. PDA medium: 200g peeled potatoes, add appropriate amount of distilled water to cook for 30min, add glucose 20g, MgSO 4 ·7H 2 O 1.5g, KH 2 PO 4 3.0g, V B1 50mg, 18g agar, distilled water to make up to 1000mL, pH natural, 115°C, 30min high temperature sterilization.

[0046] (2) Inoculate the mycelium at the edge of the activated Trichomonas nigeri colony on the VBC medium, and after culturing at 28°C for 7 days, wash the mycelia with 5 mL of sterile water to collect the spores of Tribulus nigeri.

[0047] VBC medium: potassium dihydrogen phosphate 1.0g, potassium nitrate 1.0g, sucrose 0.5g, V B1 100mg, VC 100mg, agar 20g, distilled water to 1000mL, pH natural, 115°C, 30min high temperature ste...

Embodiment 2

[0083] The gene marker of embodiment 2 black spore truffle

[0084] 1. Experimental method

[0085] 1.1 Preparation of spores of Truffle nigrosporum

[0086] Concrete method is with embodiment 1.

[0087] 1.2 Transformation and cultivation of Agrobacterium tumefaciens

[0088] Concrete method is with embodiment 1, and used plasmid is pCAMBIA1302 ( Figure 5 ).

[0089] 1.3 Co-culture and selective culture of Agrobacterium tumefaciens and spores of Truffle nigeri

[0090] Concrete method is with embodiment 1.

[0091] 1.4 Screening and identification of truffle transformants

[0092] 1.4.1 Screening of truffle transformants

[0093] Pick the single colony grown on the PDA medium to a new selective medium, isolate the hygromycin-resistant truffle strain, and keep the resistant strain in the medium containing 50 μg / mL hygromycin B Cultured for three generations until stable growth.

[0094] 1.4.2 Detection of Fluorescence Intensity of Transformants of Trichomonas nigeri ...

technical field

[0001] The invention relates to a method for transforming black truffle, in particular to a method for genetic transformation of black truffle mediated by Agrobacterium tumefaciens, and belongs to the field of genetic transformation of black truffle. Background technique

[0002] Black spore truffle (Tuber melanosporum) is an ectomycorrhizal edible fungus native to France and symbiotic with the roots of oak, oak and other trees. It is famous for its unique fragrance and "diamond" status attention. The completion of the genome sequencing of Niger truffle has promoted researchers' understanding of the biology of Niger truffle at the molecular level, but because the niger truffle has not yet established a mature genetic operating system, it greatly limits the functional genomics of Niger truffle. Research.

[0003] The Agrobacterium tumefaciens-mediated filamentous fungal transformation method developed in recent years has the following three advantages: First...

Examples

Embodiment 1

[0042] The construction of embodiment 1 black spore truffle mutant library

[0043] 1. Experimental method

[0044] 1.1 Preparation of spores of Truffle nigrosporum

[0045] (1) Inoculate the mycelia of Trichomonas nigeri on PDA medium, and culture and activate at 28°C for 3 days. PDA medium: 200g peeled potatoes, add appropriate amount of distilled water to cook for 30min, add glucose 20g, MgSO 4 ·7H 2 O 1.5g, KH 2 PO 4 3.0g, V B1 50mg, 18g agar, distilled water to make up to 1000mL, pH natural, 115°C, 30min high temperature sterilization.

[0046] (2) Inoculate the mycelium at the edge of the activated Trichomonas nigeri colony on the VBC medium, and after culturing at 28°C for 7 days, wash the mycelia with 5 mL of sterile water to collect the spores of Tribulus nigeri.

[0047] VBC medium: potassium dihydrogen phosphate 1.0g, potassium nitrate 1.0g, sucrose 0.5g, V B1 100mg, VC 100mg, agar 20g, distilled water to 1000mL, pH natural, 115°C, 30min high temperature ste...

Embodiment 2

[0083] The gene marker of embodiment 2 black spore truffle

[0084] 1. Experimental method

[0085] 1.1 Preparation of spores of Truffle nigrosporum

[0086] Concrete method is with embodiment 1.

[0087] 1.2 Transformation and cultivation of Agrobacterium tumefaciens

[0088] Concrete method is with embodiment 1, and used plasmid is pCAMBIA1302 ( Figure 5 ).

[0089] 1.3 Co-culture and selective culture of Agrobacterium tumefaciens and spores of Truffle nigeri

[0090] Concrete method is with embodiment 1.

[0091] 1.4 Screening and identification of truffle transformants

[0092] 1.4.1 Screening of truffle transformants

[0093] Pick the single colony grown on the PDA medium to a new selective medium, isolate the hygromycin-resistant truffle strain, and keep the resistant strain in the medium containing 50 μg / mL hygromycin B Cultured for three generations until stable growth.

[0094] 1.4.2 Detection of Fluorescence Intensity of Transformants of Trichomonas nigeri ...

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