Studies Of Breeding Pigment Mutant Strain Of The Steroid Hydroxylation Aspergillus Flavus By Low Energy Ion Implantation

In the modern biomedical industry, the steroid hydroxylation is one of the hotest areas of biological transformation. It has tremendous economic and social benefits. In the industry, to obtain oxygen atom, it usually use hydroxylation enzyme produced by some certain micro-organisms to add alpha- or beta- configuration hydroxyl to the C11 of steroid. Aspergillus flavus involved in this study is the strain which has very strong capacity of the steroid C11-alpha hydroxylation, but this strain will produce a red pigment during the fermentation, which is very difficult to remove with conventional ways, thereby affecting product separation and purification. This experiment uses low energy ion to mutate the original strain Aspergillus flavus , studies the parameters of implantation, has bred a mutant strain which has no red pigment produced during the fermentation and analysed its genetic characteristics, uses RAPD to analyse the difference between the two strains in molecular level, the following main results were :1. After dealing with low energy ion, the original strain shows a "saddle type" dose- survival curve; in the same implantation energy and dose, the greater quality, the lower survival rate; N+ mutation rate is higher than Ar+. To Aspergillus flavus, the best implantation ion is N+, implantation energy is 10keV and implantation dose is 1.56×1015ions/cm2~2.08×1015ions/cm2.2. Through two round screening, one strain which has no red pigment produced during the fermentation was bred. Its absorbency inλ=395nm is noticeably lower than the original strain. Further experiments show that the mutant strain has stable genetic characteristics. At the same time, thin layer chromatography to estimate the transformation rate of the steroid indicates that the hydroxylation ability of the mutant strain has not been obviously affected.3. In order to compare the differences between the starting strain and mutant strain in the molecular level, we use 102 random primers to do RAPD analysis and has found eight random primers which have different amplifications in two pools, the frequency of polymorphic primers is 7.85%. After cloning the segments which are amplified by S96,S382,S388 to analyse their sequence, we find some homologous sequences with Aspergillus fumigatus Af293 hypothetical protein (Afu6g02340),Aspergillus fumigatus Af293 3-isopropylmalate dehydratase (Afu2g11260) partial mRNA and Aspergillus nidulans FGSC A4 hypothetical protein (AN5886.2) mRNA by BLAST in NCBI, but their significance has yet to be studied further. 4. This experiment also studied the fermentation conditions for pigment generation preliminary, including fermentation time and temperature, vaccinations and the concentration of steroid, which can be references as practical industrial production.