Method for constructing aspergillus oryzae transgenic system taking phleomycin as screening marker/GFP (Green fluorescent protein) as reporter gene

Abstract

The invention provides a method for constructing an aspergillus oryzae transgenic system taking phleomycin as a screening marker / GFP (Green fluorescent protein) as a reporter gene. First, codon optimization is performed on an encoded phleomycin resistance gene acetyltransferase blmB; a blmB gene expression cassette is constructed by taking gpdA as a promoter of gene expression and trpc as a terminator; then a pEX1 carrier is transformed into a binary recombinant carrier blmB-pEX1 carrying a blmB gene complete expression nucleus and the fluorescent reporter gene GFP; the recombinant carrier blmB-pEX1 sequentially transforms GV3101 agrobacterium competent cells; then spores of aspergillus oryzae and a bacteria solution of agrobacterium are co-cultured; agrobacterium-mediated aspergillus oryzae gene transfer is performed; after co-culture, screening is performed through a phleomycin resistance screening culture medium; finally, resistant transformants are picked for expanded culture, anda fluorescent microscope is used to observe the successful expression of the GFP. The false positive rate of the transformants is effectively reduced, the screening workload is reduced, and the problem of insufficient resistance screening markers is solved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing an Aspergillus oryzae transgenic system using phleomycin as a selection marker / GFP as a reporter gene. Background technique [0002] Aspergillus oryzae belongs to the genus Aspergillus and is a strain capable of producing compound enzymes. The enzymes it produces include protease, amylase, glucoamylase, cellulase, phytase, etc. Fermentation industries such as brewing play a very important role, and are widely used in the production of soy sauce, rice wine, commercial enzymes and medical proteins. Using Aspergillus oryzae as the production strain, it has mature fermentation and post-processing technology, so it is often used as an ideal tool for gene expression, protein secretion research and heterologous protein expression. However, in actual production, the enzyme-producing ability of Aspergillus oryzae is often not good. By genetically engin...

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Problems solved by technology

[0004] Aspergillus oryzae is a kind of filamentous fungus, which is resistant to various antibiotics and fungicides. There are few types of drug resistance markers reported. At present, there are only pyridinethiamine resistance genes encoded by ptrA and AoadhB The encoded wiltingin resistance gene has been applied in the transgenic system of Aspergillus oryzae (Suzuki et al., 2009). Although the effect of these two screening markers is good, each screening marker has its advantages and disadvantages. The high price of thiamine limits its use, so the development of more resistance selection markers for Aspergillus oryzae will provide an effective way for transgenic manipulation of Aspergillus oryzae

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