Expression And Function Research Of Five Cysteine Proteases From Populus Trichocarpa Torr.& Gray

Cysteine protease is a large of class of protease family,which is widely involved in the programmed cell death such as protein hydrolysis, leaf senescence, and so on.The study selected Populus trichocarpa as experimental material and analyzed the tissue expression, protein characteristics and the germination and development of transgenic Arabidopsis thaliana under drought stress of five cysteine proteases by protein structural analysis, real-time PCR, prokaryotic expression and transgenic technology.In this study, PtCP1, PtCP3, PtCP4 and PtCP5 were mainly analyzed by bioinformatics.The signal peptide sequences were analyzed by SignalP3.0 software;the isoelectric point, molecular weight and other factors were analyzed by DNAMAN software;the amino acid sequences were compared and analyzed by BioEdit software.The result showed that PtCP2 and PtCP4 were members of the RD19A-like of papain and PtCP3 and PtCP5 were members of the CTB3-like of papain.PtCP1, PtCP3, PtCP4 and PtCPS were analyzed of tissue expression and localization by real-time PCR in this study.Real-time PCR results showed the PtCP2, PtCP1, PtCP4 and PtCP5 were mainly expressed in root and leave but had low expression level in stem of Populus trichocarpa.The result indicated that the function of cysteine protease gene family may be related to drought stress.The expression of PtCP2, PtCP3, PtCP4 and PtCP5 were induced in vitro by prokaryotic expression and the recombinant proteins were purified by washing method on inclusion body.PtCP2, PtCP3, PtCP4 and PtCP5 could be induced by IPTG in prokaryotic expression system and the proteins were weighed as 38.2 kD,36.7kD,38kD and 36.7kD respectively after removal of the signal peptide.The purified recombinant proteins could be obtained after purifying by washing method on inclusion body.Proenzyme proteins attempted to activate by autocatalysis in acidic conditions but it didn’t work.The result showed that the five cysteine protease zymogens may not be activated by autocatalysis but by other protease.The expression vectors pB1121-PtCP1, pBI21-PtCP2, pBI121-PtCP3, pBI121-PtCP4 and pB1121-PtCP5 were constructed and transformed into wild-type Arabidopsis thaliana using floral dip method and then the PtCP1 and PtCP5 transgenic Arabidopsis thaliana lines were obtained.To test the function of PtCP5,the transgenic lines were analyzed in germination rate and length of root under normal conditions and drought stress respecitively.The results of analysis in PtCPS transgenic Arabidopsis thaliana showed that PtCP5 had a positive effect on the root growth and it can also increase the tolerance to drought stress in Arabidopsis thaliana.The research is of great significance to understand the expression and function of cysteine protease in woody plants deeply and will lay the foundation for the further study of molecular mechanism of cysteine protease family in growth and development of woody plants.