The Studies On Aoz1 Gene And Its Promoter

In recent years,the misuse of chemical anthelmintics is very common,which has caused some problems on control of parasitic nematodes and brought immeasurable economic losses to the animal husbandry.Therefore,it is very necessary to develop a novel and safe method for treating domestic nematodiasis.Fortunately,the natural enemies of nematodes,the nematode-trapping fungi,have been finally discovered through unremitting efforts of relevant scholars and shown good application prospects for the prevention and control of domestic animal nematodiasis.After years of research by scholars,it has been found that nematode-trapping fungi secrete a variety of extracellular proteases,including serine proteases working during the invasion and digestion of nematodes.In order to ascertain more information of the Aoz1 gene and its promoter,to further explain the role of in predation of nematode-destroying fungi against parasitic nematodes,the study on the Aoz1 gene of Arthrobotrys oligospora and searching for the gene promoter were carried out.The research process and results are as follows:(1)Firstly,the coding sequence of serine protease of Arthrobotrys oligospora was amplified and constructed into prokaryotic expression vector-pET32 a,then it was transfered into Transetta(DE3)expression cells.Latter,the expression conditions were optimized and the product was purified.The results showed that the serine protease Aoz1 gene of Inner Mongolian strain of Arthrobotrys oligospora can be expressed in the Transetta(DE3)expression cells,and its fusion expression product existed in the form of inclusion bodies.Its optimal IPTG induction concentration was 1.5 mmol/L and the highest production was got post 8h incubation.Purification of the recombinant protein was performed finally.(2)Based on the published genome sequence and serine protease gene(Aoz1)of Arthrobotrys oligospora,BLAST was used to compare the two sequences to find the Aoz1 gene upstream sequence and design primers for amplification and cloning this sequence.Then the sequence was analyzed by the promoter online software Promoter Scan,NNPP,Promoter 2.0,and the CpG island was also predicted with online software EMBOSS and Meth Primer.The results showed that the length of the obtained sequence was 2009 bp,and the homology with the original sequence was 96%;There were TATA box,transcription start site TSS,transcription factor binding site and CpG island in the sequence of 2009 bp upstream of the start codon of the extracellular serine protease Aoz1 of Arthrobotrys oligospora.(3)Recombinant of firefly luciferase reporter gene vector of pGL3-Basic-P1 and5’-end deletion recombinant vectors,pGL3-Basic-P2 and pGL3-Basic-P3,were constructed and transfected into HEK-293 T cells and HeLa cells,respectively.Then the activity of the luciferase were detected.The results showed that the 2009 bp upstream of the start codon of the Aoz1 gene can significantly increase the expression activity of the firefly luciferase reporter gene in HEK-293 T cells and HeLa cells.The core promoter region of the Aoz1 gene may be located in in the sequence P1,which could supply the crictical data for the study of extracellular serine protease Aoz1 gene and the trapping mechanism of the Arthrobotrys oligospora.