Differential Studies On The Expression Of Rice OsAPX Family Genes Under Alkaline-saline Stress

When confronted drought, salt and alkali, high-temperature, cold, and water stress, the dynamic balance between the generation and scavenging of free radicals and reactive oxygen species (ROS) in plant cells will be disrupted, which will induce oxidative stress of plant, restrict its growth and productivity, even the death of plant. Ascorbate peroxidase (APX, EC1. 11. 1. 11) is one of the main enzyme that scavenging H₂O₂, which is a facter gernerating oxidative stress, through catalyzing H₂O₂ deoxidized to H₂O in higher biology. Recent study had showen that salt and alkali stress elicite excess acumμtlation of H₂O₂ in plant cell which may lead to oxidative stress. Whether over-expression of enhanced APX ROS scavenge system can improve salt and alkali tolerance of plant or not is worthy of investigating. APX is a multigene family in plant. Genomic analysis of rice indicated that OsAPX gene family has three types including eight members, that are: cytoplasm APX, OsAPX1, 2; peroxisome APX, OsAPX3, 4; chloroplast APX, OsAPX5, 6, 7, 8. In order to clarify the difference in expression character of OsAPX gene family under salt and alkali stress, also the difference in salt and alkali stress tolerance improved by over-expression of different APX gene members, we selected six members of OsAPX gene family that submitted to three types, investigated thesis above, based on bioinformatic analysis. The results were as followed:1. The difference in expression character of six members of OsAPX gene family was comparative analyzed under stresses. Northern blot analysis indicated that, the transriptional expression of OsAPX1, 2, 3, 4, 7 gene was increased both in leaf and in root with the time prolonging when exposed to 100mmol/L NaCl, 20mmol/L NaHCO₃ and 10%PEG6000 stresses. Esbecially OsAPX1, 2, 4, 7 were more prone to response to stress. Further analysis indicated that OsAPX1, 2, 4 transcript levels were increased alter stressed by 10% H₂O₂, 100mmol/L NaCl, 20mmol/L NaHCO₃, 20mmol/L Na₂CO₃ and 10%PEG6000 for 24h, OsAPX2, 4 increased significantly after exposed to H₂O₂ and salt stress. In order to clarify whether the signifcant increasing of OsAPX genes was caused by H₂O₂ generated during salt and alkali stress, transcript expression character of OsAPX2, 4 was determind by Northern blot analysis, with OsAPX2, 4 as diversity probes. With the increasing of concentration, OsAPX2, 4 transcript expression increased after stressed by H₂O₂ for 24h, and the increasing range of OsAPX4 was larger than that of OsAPX2. At the same time, the transcript expression character of OsAPX4 and OsAPX7 were analyzed under heavy metal stress of 100μmol/L CdCl₂, ZnCl₂ and CuCl₂. The resμlt indicated that OsAPX4 is higher than OsAPX7 in roots. To validate the relationship between the transcript expression of OsAPX gene and activity of APX, the stress-induced changes of APX isoenzymes in different organs (leaf, root, stem, leaf sheath) of rice were assayed. It was obviously that total activity of APX in root was increased significantly under salt and alkali stress. For the sake of further investigating the changes of OsAPX gene on protein expression level under stresses, expression and purification of fusion GST-OsAPX2 protein in vitro was achieved using an Escherichia coli expression system. Based on this, polyclone antibody of OsAPX2 was purified by immunity rabbits. Western blot analysis of rice protein confirmed that APX isoenzyme is a multi-gene family. APX protein was induced and higher than controls in rice seedlings exposed to salt and alkali (NaHCO₃,Na₂CO₃, NaCl) and H₂O₂ treatment both in leaf and root. For CdCl₂ stress, however, this trend was only observed in root.2. In order to compare the salt and alkali tolerance of two cytoplasm OsAPX genes (OsAPX1, 2), OsAPX1, 2 was introduced into yeast, Arabidopsis thaliana (ecotype: Columbia) and tobacco (Nicotiana tabacum cv. K₃₂₆), respectively. Alkalinity-salinity tolerance identification indicated that yeasts recombined OsAPX2 gene was more tolerant than that of recombined OsAPX1 gene. This phenomenon was also found in the T₃ transformants of Arabidopsis stressed by 150mM NaCl or 5-10raM NaHCO₃. Moreover, both OsAPX2 and OsAPX1 transformants were more tolerant to salt and alkali stress than wild controls. Further alkalinity-salinity tolerance identification of potted transformants also proved this. Oxidation damage tolerance of transformants leaf was measured by vigorous oxidant Na₂SO₃ and H₂O₂, with the result following the order of OsAPX2 transformants＞OsAPX1 transformants＞wild type controls. The OsAPX1, 2 gene was introduced into tobacco by Agrobaterium mediated transformation. Southern blot analysis indicated that OsAPX1, 2 is mono-copied into tobacco genome respectively, while Northern blot analysis confirmed its transcript expression. T₂ transformants over-expression OsAPX1, 2 gene promoted by CaMV35S promoter was tolerant to salt and alkali stress. Besides, under certain concentrations, OsAPX2 transformants were more tolerant to NaHCO₃ stress than OsAPX1 transformants. Oxidation damage tolerance of T₂ transformants leaf was measured by vigorous oxidant Na₂SO₃, H₂O₂ and Paraquat, with the result showed that OsAPX2 transformants were more tolerant to Oxidation damage than OsAPX1 transformants. The stress-induced changes of APX isoenzymes in T₂ transformants indicated that total activity of APX was increased in both transformants, and the increasing range of OsAPX2 transformants was larger than that of OsAPX1 transformants. This verified the difference between two cytoplasm OsAPX genes (OsAPX 1, 2) in salt and alkali tolerance.3. The difference on salt and alkali tolerance between two peroxisome OsAPX genes (OsAPX3, 4) were further investigated in yeasts and Arabidopsis transformants. When exposed to NaCl and NaHCO₃ stress, the germinating status of Arabidopsis transformed OsAPX4 gene was significantly better than that of transformed OsAPX4 gene, implying that alkalinitysalinity tolerance of Arabidopsis transformants improved by over-expressing OsAPX4 gene was higher than that of over-expressing OsAPX3 gene. The succeed in Expression and purification of fusion GST-OsAPX3, 4 protein in vitro using an Escherichia coli expression system paved the way for specific activities analysis of isoenzyme and preparation of antibody next step. Alkalinity-salinity tolerance assay of yeasts transformed OsAPX4 gene indicated that transformants grown better than wild type controls. Compared with wild type controls, T₃ transformants of Arabidopsis plants transformed OsAPX4 gene were more tolerant to 200 mmol/L NaCl stress for 15 days and 7.5mmol/L NaHCO₃ stress for 12 days, respectively. OsAPX4 gene may play an important role in surviving of yeast and Arabidopsis transformants under salt and alkali stress.4. For tolerant breeding of transgenic alfalfa, the OsAPX2 gene was introduced into callus of cotyledon of alfalfa (Medicago sativa L.) using an Agrobacter-mediated system. The regenerated plantlets were obtained through Kana differentiation and PCR determination with specific primers. Northen blot determination of T₂ plantlines confirmed that the rice OSAPX₂ was integrated into alfalfa, and the average activity of APX of T₂ plantlines enhanced to 3. 15- fold than that of wild type controls. Alkalinity-salinity tolerance of transgenic lines needs to be further studied.