嗜线虫致病杆菌HB310菌株pirA和pirB基因的克隆表达及特性的研究-农业昆虫与害虫防治专业论文.docxVIP

Cloning，expression Cloning，expression and characteristics of thepirA and pirB gene from Xenorhabdus nematophila HB310 Candidate：Zhang Jie Advisor：Professor Wang Qin—ying Speciality：Agricultural Insect and Pest Control Abstract Xenorhabdus nematophila HB3 1 0 form Steinernema carpocapsae HB3 1 0，iS symbiotic bacteria with a wide spectrum of insecticidal activities and antifungal activities． PirB protein was isolated from intracell total proteins by method of native—PAGE，and identified by mass spectrometry analysis．PirB protein showed a high hemocoel injecting activity(LDs0=1 3．92ng／larvae)against the 5m instar larvae of Galleria mellonella． We also found the pirA gene near the pirB gene in the X nematophila genome sequence．In order to further understand the characters of pirB gene and its relationship with pirA gene．both pirA gene and pirB gene were cloned from X nematophila HB3 1 0 DNA，and sequenced through constructing the clone vectors of pMD 1 8一T-pirA and pMD 1 8-T-pir8．The results of bioinformatics analysis showed that pirA had an open reading flame(ORF)of 408bp，coding 1 3 5 amino acid．The deduced protein PirA was a stable water-soluble protein with a 14．9kDa molecular weight，5．0 isoelectric point and no transmembrane domain．There is a probability of 82．6％for PirA to be located in the nuclei． Ⅵmile pirB had an ORF of 1 290bp．coding a stable water-soluble protein of 429 amino acid，48．2kDa，5．0 isoelectric point with transmembrane domain but no signal peptide． PirB shared the same domains with borth endotoxin and a lectin of Jacalin．PirB had a probability of 69．6％to be located in nuclei，21．7％to in mitochondria and 8．7％in cytoplasm．The secondary structure predictions showed that PirA had 1 2 13-pleated sheets but no 0【．helix．while PirB had 8 0【．helix and 1 2 13-pleated sheet．At last，tlle tertiary structure of the two proteins were predicted by homology modeling． In order to clarify the activity and relationship of PirA and PirB，the prokaryotic expression vectors