Cloning Of Glutathione Reductase In Eucalyptus Grandis×E.ophylla And Analysis Of Expression Pattern Under Low Temperature

The copper chaperone for superoxide dismutase(Cu/Zn SOD,CSD)is composed of copper chaperone for superoxide dismutase(SOD)dependent CCS and non-dependent CCS activated pathway,which plays an anti-oxidative detoxification effect.Glutathione reductase(GR)catalyzing the conversion of glutathione to glutathione(GSH)may transport copper ions to SOD and activate it,which may associated with a non-CCS activated pathway for plant cytoplasmic CSD.In this study,a new gene containing 1485 bp open reading frame and encoding 495 amino acids in Eucalyptus was cloned by RT-PCR technology,which named as Eu GR(Gen Bank Accession Number: KU904639)and located in the cytoplasm of the plant.The prokaryotic expression vector of Eu GR named p ET-Eu GR was constructed firstly and which could expressed stably with 0.1m M IPTG for 4h at 37 ?.Finally,the activity of GR in the transformed strain p ET-Eu GR was higher than that in the control strain by our test,which indicates the Eu GR has a biological activity.In addition,the results of fluorescence quantitative PCR showed that the Eu GR expressed decreased with the leaf senescence in the leaves of Eucalyptus greenhouse plants,which in young leaves is highest,Eu GR expressed most in the stem which followed by leaf and roots in the tissue culture of Eucalyptus urophylla.To explore the relationship between GR and non-CCS activating pathways of CSD1,the activity of SOD in p BI-e SOD?p BD-anti-4CL-e CSD1 transgenic Eucalyptus grandis × E.ophylla plants were determined.A comprehensive analysis based on a study related to cold-resistance ability of transgenic plants was conducted,in which six transgenic plants were selected to analyzed the expression of GR,CSD1 and CCS at room temperature,4? for 36 h,4? for48 h by fluorescence quantitative PCR,which revealed that :(1)The expression of Eu GR,Eu CCS and Eu CSD were higher in the plants with the strong cold tolerance(like P40,P41,P52)than that in plants with weak cold tolerance(like P36,F44,F76),which proved the cold tolerance of transgenic plants affected expression of GR,CCS and CSD.(2)When tansgenic plants were treated continuously at low temperature,the expression of CSD increased firstly and then decreased,the expression of Eu CSD and Eu CCD in the cold resistant plants were significantly different at the low temperature,which indicated that CCS and CSD are relate to plant cold tolerance,but it's not enough to prove they relate to the recovery ability of transgenic plants after cold injury.(3)The activity of SOD in the transgenic plants was correlated with the activation of GR and CSD1,the results showes that CSD1 could alleviate the damage caused by low temperature stress,and CCS wasgradually restored with the recovery of SOD activity after low temperature treatment,which confirmed that CCS is closely related to the activation of CSD.(4)The expression of GR gene decrease continuously at low temperature for 36 h,when the expression level of GR gene was significantly higher than that of CCS,we think the non-CCS pathway of CSD1,which participates with other factors of action under low temperature stress,may dominate the activating pathway of CSD1.