Secretion Expression of Brassica napus pgip Gene Removing Singal Peptide in Pichia pastoris

doi:10.7668/hbnxb.2015.06.004

Abstract

Abstract: In this study,according to the sequence of pgip9,pgip15 CDS in GenBank,the primers were designed,and removed the signal peptide sequence,the total RNA of rapeseed leaves inoculated with Leptosphaeria biglobosa (NM-1) after 7 days,were extracted and reversed transcription cDNA as template.The pgip9 and pgip15 coding region fragments were amplified excluding signal peptide by PCR,and cloned into Pichia pastoris expression vector constructing the recombinant plasmid pPICZαA- pgip9 ,pPICZαA- pgip15.The recombinant plasmids were identified by single,double enzyme digestion,colony PCR,and then were transformed into yeast cells PMAD16,then were identified by YPD including Zeocin^TM and colony PCR inducing by methanol,analyzed by SDS-PAGE and Western Blot,producing a single band of approximately 36-37 kDa,proteins were expressed and secreted successfully.The expression of PGIPs in vitro were conducted to provide a basis for the interaction between PGIP and PG,and the pgip gene function in canola resistant to phoma stem canker.

Key words: Leptosphaeria biglobosa, pgip, Deleted singal peptide, Pichia pastoris, Secretion expression

CLC Number:

S565.4
Q78

HUANGFU Hai-yan, YAN Meng-jiao, SONG Pei-ling, HAO Li-fen, HUANGFU Jiu-ru, JIA Xiao-qing, LI Zi-qin. Secretion Expression of Brassica napus pgip Gene Removing Singal Peptide in Pichia pastoris[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(6): 27-31. doi: 10.7668/hbnxb.2015.06.004.

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