Cloning And Expression Of Spinacia Oleracea Nitrite Reductase In Pichia Pastoris

Nitrite is a kind of widespread chemicals in the nature，especially in food.Including meat,fish, rice, eggs, vegetables. Vegetable is one of the most popular food intaked by people. It iseasy to enrichment of nitrite in vegetables,80%~90%of the nitrite in the body was intakedfrom eating vegetables, especially from leafy vegetables.The studying of the NIR gene cloningof spinach, its expression and biological analysis is very useful for harm reduction of nitrite onthe humanbody and it has theoretical significance and important application prospect.In this paper using molecular biology methods, according to GenBank (X07568) registeredOpen Reading Frames of Reductase Spinacia, and the ends of the open reading frame usingprimers design principles synthesize the corresponding of primers.In the study using rhe methodof RT-PCR can get the RNA of NiR spinach and verify the integrity RNA of NiR spinach.By theFirst Stand of cDNA,reverse the RNA of spinach NiR transcribed into cDNA fragment,then itwas constructed pPICZα A. Recombination expression vector pPICZα A-nir was constructed,and successfully cloned target gene.Compared the size of the plasmid vector,double digestionand the PCR amplification of identification,It is confirmed that the expression vectors are theSpinacia Oleracea Nitrite reductase.Protein expression system of the spinach NiR is Pichia pastoris GS115,in the course of theexpression. Expression vector is linearlized by Sac I, and is transformed into Pichia pastorisGS115though electroporation.So that the extracted of the gene is connected to expressionvector.Cloning of NiR spinach gene by using the RT-PCR method, and using ExPASy analysis topredict its coding protein. The result proved that, the long of NiR gene was2062bp,contained1785bp Open Reading Frames. Encoding a594amino acid residues of the polypeptide chain.Amino acid sequence alignment analysis of the display, the amino acid sequence and both theamino acid sequence of wheat code and Arabidopsis thaliana has85%homology.Containing a crude enzyme solution of spinach nitrite reductase gene,according to the Griessreagent colorimetric method was used to determine the nitrite content, the enzyme activity wasmeasured266.18U/mL,the extracted bacterial liquid has higher enzyme activity. In this paper，using diazotized coupling reactions,5uL of nitrite bacteria were stained by nitrite solidmedium.The result showed colorless can contain bacteria of spinach nitrite reductase activity ishigh,nitrite of spinach has a strong ability to degrade.In summary, This study constructed successfully pPICZa A-nir and was transformed into Pichia host cell GS115, the recombinant strain GS115-nir. Through comparing the nitritereductase, walnut, garlic, ginger on nitrite degradation rate was found to have varying degrees ofdegradation,The results showed that the cleaning rate Spinacia Oleracea Nitrite reductase were42.80%,walnut juice were15.31%, garlic juice were36.85%, ginger juice were18.5%. Resultsshowed that the cleaning rate Spinacia Oleracea Nitrite reductase were better than others.