Tissue Specific Expression And Function Analysis Of Cysteine Protease PtCP5 From Populus Trichocarpa Torr.&Gray

Cysteine protease is an important type Protease family that participates in various plant physiological processes such as low temperature,drought,salt stress,plant immunity,seed germination and programmed cell death(PCD).The PtCP5(Cysteine Protease 5)gene of Populus trichocarpa was cloned in previous research of the laboratory.Now,the PtCP5 gene promoter fragment was cloned,and analyzed.The cis-acting elements in promoter fragment,tissue localization expression,and subcellular localization were also analyzed.In addition,the phenotypic characteristics of transgenic PtCP5 Arabidopsis thaliana were analyzed under normal and stress conditions,so as to explore the possible biological functions of PtCP5.The main results are as follows:1.The 1372bp promoter fragment of the cysteine protease PtCP5 gene was cloned.These cis-acting elements,such as G-box element responses light regulation,ABRE element responds to ABA,Aux RR-core element responds to IAA auxin,TATC-box element responds to gibberellin,TGACG-motif and CGTCA-motif elements respond to methyl jasmonate,MYB and MYC elements respond to drought and ABA,were related with light response,abiotic stress response,hormone response,and growth and development response,indicating that PtCP5 may participate in the regulation of multiple stress signal responses,and play a key role in the growth and development of Populus trichocarpa.2.The vector ProPtCP5-GUS-p BI121 with the PtCP5 self-promoter connected to the GUS gene was constructed and transformed into Arabidopsis thaliana.The GUS activity was detected in root,stem,cotyledon,flower and other parts.Moreover,the 35S-PtCP5-GFP vector was constructed and transformed into Nicotiana tabacum.Transient expression analysis revealed that PtCP5 localized in cytoplasm.It is speculated that PtCP5 gene may be related to protein mobilization and degradation.3.The phenotype of PtCP5 transgenic Arabidopsis was analyzed.The results showed(1)Comparing with wild-type(WT),the germination of PtCP5 transgenic seeds was delayed.The coat thickness and protein types of transgenic PtCP5 and WT seeds were no significant differences.The storage protein content of PtCP5 transgenic seeds is significantly lower than WT,indicating that PtCP5 participates in the mobilization of storage proteins in seeds,and the premature mobilization of stored proteins in overexpressed PtCP5 may be the cause of slowly seed germination.(2)The transgenic PtCP5 Arabidopsis flowers were normal,while most of the transgenic PtCP5 Arabidopsis pollen was abnormal,suggesting that the overexpression of PtCP5 caused pollen damage.(3)The germination rate of PtCP5 transgenic seed was significantly lower than untreated seeds during the drought stress simulated by mannitol or the salt stress simulated by Na Cl,indicating that the overexpression of PtCP5 caused abnormal seeds and poor ability of drought and salt tolerance.4.The strain containing the recombinant expression vector p ET30a-?VPE,p ET30a-PtCP1 and p ET30(+)-PtCP5 which were constructed by previous research of the laboratory were induced by IPTG for prokaryotic expression in vitro.Recombinant protein At?VPE,PtCP1 and PtCP5 were in the form of inclusion bodies,and soluble proteins were obtained by further purification.At?VPE was used to activate PtCP1 or PtCP5 in vitro,but failed to obtain mature PtCP1 or PtCP5 proteins,indicating that At?VPE may not be able to transform and activate PtCP1 or PtCP5 precursor proteins in vitro,cysteine proteases PtCP1 and PtCP5 may be activated by other proteases.In this study,promoter of the cysteine protease gene PtCP5 was cloned and expression location was analyzed analysis.The possible biological function of PtCP5 was also explored.It is very significance to reveal the function of the cysteine protease family in woody plants.