Cloning And Characterization Of The Antioxidative Genes In Spartina Auerniflora

Spartina alterniflora is a highly salt-tolerant halophyte, in which unique resilience mechanism may exist. The mechanism of releasing active oxygen species may also be different from that in other species. Therefore, cloning and functional analysis of genes regulating active oxygen scavenging enzyme in halophytes S. alterniflora may help us to understand the mechanism of oxidative stress tolerance in plants.In this work, the activities of O2· and H2O2eliminating enzymes, SOD, CAT and APX in S. alterniflora are investigated systematically with high NaCl concentration treatments. Their roles in salt tolerance are clarified in combination with other physiological indicators in this species. To gain a better understanding of the relationship between salt tolerance and ROS scavenging system, and to shed more light on the correlation between the tolerance and antioxidants in halophytes, I clone APX1, APX11and CAT1genes in S. alterniflora and study their roles under oxidative stress caused by high salt concentration, heavy metals, ABA, drought, chilling and heat stresses. The results are as follows:(1) Changes of MDA (malondialdehyde) content and membrane permeability in S. alterniflora leaves under stress conditionsCompared with the controls, both MDA concentration and EL of S. alterniflora seedlings increased when treated with600mmol/L NaCl and500μmol/L Cd2+. This indicates that high salt and heavy metal Cd2+stress caused damage to the membrane system of S. alterniflora seedlings. More serious damage caused by heavy metal Cd2+is observed than that of high salt concentration.(2) Changes of proline and soluble sugar contents in S. alterniflora under stress conditionsAfter treatment with600mmol/L NaCl and500μmol/L Cd stress, proline content of the leaves significantly increased compared to that of the controls (P<0.01). The soluble sugar content also increased significantly (P<0.01) at the begining and then slowly declined. This means that proline and soluble sugar may regulate osmotic stress caused by salt stress, while proline is a major osmotica under osmotic stress caused by Cd stress in S. alterniflora.(3) Changes of O2· and H2O2contents in S. alterniflora leaves under stress conditionsTreatments with both600mmol/L NaCl and and500umol/L Cd significantly increased the O2· and H2O2contents in S. alterniflora leaves, at P<0.01and P<0.05levels, respectively. This indicates that under oxidative stresses caused by high salt and heavy metal stresses, Salterniflora produced large amout of ROS.(4) Antioxidase activity of S. alterniflora leaves under stressesSOD, CAT and APX are the most basic enzymes eliminating O2· and H2O2in higher plant. After salt treatment, the activity of these enzymes changed at different levels. The activity of SOD, which reducts O2· to H2O2, is significantly higher than that of the controls. The enzyme activity rose rapidly at early stages and then decreased as the treatment time extended. The activities of the H2O2eliminating enzymes, CAT and APX, are significantly higher than those of the controls (P<0.01). While the salt treatment induces the same changes as those of SOD, the Cd treatment induces a continued significantly rise of enzyme activity. Result from non-denaturing polyacrylamide gel electrophoresis shows that the overall changes of the enzyme activities agreed with the above measurements, although isoenzymes in different subtypes had slightly different trends over time. These results imply that S. alterniflora may have a strong ability of resistance to oxidative stress caused by salt stress and heavy metal stress.(5) Cloning and sequence analysis of SaAPX1、SaAPX11and SaCAT1genes in S. alternifloraThree antioxidant genes, SaAPX1, SaAPX11and SaCAT1are cloned from S. alterniflora using degenerate PCR,5’RACE and3’RACE methods, All these genes encode cytoplasmic proteins. SaAPX1and SaAPX11sequences are highly similar, with only a small number of nucleotide differences in the C-terminus.(6) Expression profiles of SaAPXl, SaAPXll and SaCAT1genes in S. alternifloraResults show that the expression levels of SaAPXl, SaAPXll and SaCATl increased in all treatments, including20%PEG,100μmol/L ABA, chilling and heat, with minor variations in expression profiles among treatments..(7) Function studies of SaAPXl, SaAPX11and SaCAT1genesScreening for Rice and Arabidopsis homozygous strains overexpressing SaAPX1, SaAPX11and SaCAT1is currently underway, while physiological and biochemical analysis are to be carried out.