It was expected that recombinant Aspergillus niger glucose oxidase could be expressed in Trichoderma reesei with stable activity. T. reesei CBHI promoter--CBHI ss. gene--A. niger glucose oxidase gene--T. reesei CBHI terminator--A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator--pUC19 (pCBHGOD) vector was constructed in E. coli DH5alpha by PCR application and gene cloning methods. T. reesei QM9414 protoplast was transformed by T. reesei CBHI promoter-CBHI ss. Gene--A. niger glucose oxidase gene--T. reesei CBHI terminator-A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator linear DNA fragment (CBHGOD fragment) that was made by digestion of pCBHGOD with Kpn I. T. reesei mutant clone with homologous recombinant A. niger glucose oxidase gene was selected by PCR method. Recombinant glucose oxidase was produced by mutant T. reesei strain under induction of wheat straw for 5 days. Recombinant glucose oxidase molecular mass was showed the same as native A. niger glucose oxidase standard from Sigma company by Western blot analysis. Recombinant glucose oxidase activity was 25u/mL in medium. The yield was 0.5 g/L in comparison with Sigma company glucose oxidase standard. There was no recombinant GOD degradation during Trichoderma reesei cultivation that was showed in Western blot analysis. Trichoderma reesei has capability to be a new recombinant host for Aspergillus niger GOD production.
Aspergillus niger ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Glucose Oxidase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Trichoderma ; genetics ; metabolism

AIMTo investigate the change of NMDAR1 (zeta 1) subunit expression in temple cortex, frontal lobe, hippocampus and cerebellum of three different group rat after 98 dB wide frequency noise exposure.

METHODSWestern Blot and RT-PCR technique, combined with auditory brainstem response (ABR) measurement.

RESULTS(1) Expressions of NMDAR1 (zeta 1) subunit in frontal cortex, temple cortex, hippocampus and cerebellum have no difference, but AD model rat is much weaker than the control group. (2) Expression of NMDAR2A (epsilon 1) in temple cortex for physiological saline groups rat have a mostly increase (plus noise), moreover, those are weakest expression in hippocampus. NMDAR1 (zeta 1) subunit in cerebellum have highest expression, moreover, it is weakest in temple cortex. (3) NMDAR1 (zeta1), NMDAR2A (epsilon 1) subunit expression in hippocampus for three groups rat have a down-regulation after adding noise. (4) NMDAR1 (zeta 1), NMDAR2A (epsilon 1) subunit mRNA expression in control group have no remarkable difference in different cortex. (5) Expressions of NMDAR2A (epsilon 1) in frontal temple cortex, hippocampus for AD model rat are less than that of other groups, weakest in cerebellum, weaker in frontal.

CONCLUSIONWide band frequency noise can reduce the expression of NMDAR1 (zeta 1) subunit in hippocampus and cerebellum of AD model rat, however, the way of regulation is not in the mRNA level. Wide band frequency noise can inhibit the expression of NMDAR2A (epsilon 1) in hippocampus, temple cortex of AD model rat, which has been regulated by mRNA level and have cortex area difference.

Animals ; Brain ; metabolism ; Cerebral Cortex ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Glutamic Acid ; poisoning ; Noise ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; metabolism