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Study on the Transformation of Filamentous Fungi Mediated by Agrobacterium Tumefaciens and the Expression of Lipase Gene

Author: LiJunXing
Tutor: PanLi
School: South China University of Technology
Course: Microbiology
Keywords: Filamentous fungi Agrobacterium tumefaciens -mediated transformation Lipase SH-1 , Aspergillus niger
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 119
Quote: 1
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Abstract


Filamentous fungi are widely used in the production of recombinant proteins is being more and more attention. Receptor as genetic engineering bacteria, filamentous fungi to bacteria, yeast, and the type of unique advantages: they not only has a strong ability of protein secretion, and more similar to higher eukaryotes, it is possible to correct various translation processing. Therefore, filamentous fungi not only in the field of industrial enzyme production in the field of bio-medicine, food safety, biological and bio-pathology have a wide range of applications. With the deepening development of genetic engineering technology, more and more filamentous fungi through genetic transformation as exogenous protein expression host used in industrial production and other fields. Therefore, as one of the most critical technology - genetic transformation and its role has become increasingly important. In this study, the wild-type Aspergillus host selection marker test, hygromycin B, benomyl and pyridine thiamine drugs and the acetamide nitrogen source utilization medium for eight industrial Aspergillus growth. The results show that, the hygromycin B benomyl and pyridine thiamine for the growth of Aspergillus niger SH-1 wild-type strain and awamori, Aspergillus CBS115.52 is wild strain has good inhibition, the optimal concentration of 100ug/ml 1.5μg/mL 0.1μg/mL and poor inhibitory effects the Aspergillus oryzae IFO4177 and Aspergillus parasiticus CICC2312 of minority Aspergillus. Acetamide as the sole nitrogen source medium plates, Aspergillus niger and Aspergillus awamori 8 bacteria growth is very slow, the inhibitory effect is also good. Gateway cloning technology to build the binary expression vector containing benomyl resistance gene and the amdS gene, and host bacterium Agrobacterium tumefaciens-mediated transformation method selection, SH-1, Aspergillus niger and Aspergillus awamori CBS115.52 as completion of the above 4 kinds of the selection marker gene genetic transformation, the results show that the above marker genes have been transformed into the genome of Aspergillus niger and Aspergillus awamori, and thereby create the glucoamylase industrial production of wild-type strains of bacteria, SH-1, Aspergillus niger, Agrobacterium-mediated transformation method. On this basis, the restructuring meters Hagan Mucor lipase gene is transferred to Aspergillus niger in SH-1 from the DNA and protein levels were tested transformants screened stable genetic transformants and its expression lipase enzyme live a preliminary fermentation optimization determination.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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