Functionalanalysis Of Alfalfa MsCAMTA2 Gene In Abiotic Stress

External environment,such as salt,saline,drought and cold are the main abiotic stress factors that limit the growth and development of plant.Calcium play an important role in abiotic stress response signaling pathway.Calcium signal is induced by environmental stimulate,influxed Calcium binds to calmodulin（Ca M）and activated calmodulin binds to transcription activator CAMTA to initiate defense system.In this study,alfalfa Ms CAMTA2 gene was over-expression in tobacco by Agrobacterium-mediated transformation,analysis of the function Ms CAMTA2 under abiotic stress.The main results are as follows:1.Tissue-specific expression and expression patterns under abiotic stresses of Ms CAMTA2 in alfalfaThe results of q RT-PCR analysis showed that alfalfa Ms CAMTA2 gene was expressed in roots,stems,leaves,flowers and fruits of alfalfa,and presented highest expression in roots.Four week old alfalfa was used for stress response expression pattern analysis,under salt（200 mmol/L Na Cl）,mixed saline（100 mmol/L Na HCO₃:Na₂CO₃=2:1）,ABA（30μmol/L）and mannitol（150 mmol/L）simulated drought stress.These results showed that Ms CAMTA2 gene was induced such abiotic stresses,especially under drought stress.The expression of Ms CAMTA2 performed high increased at 1 h under 150 mmol/L mannitol treatment,presented a rapidly response to drought stress.2.Genetic transformation of alfalfa Ms CAMTA2 gene to tobaccoThe Ms CAMTA2 gene was transformed into tobacco by Agrobacterium-mediated method.Totally 32 transgenic lines were screened by PPT.20 transgenic tobacco lines were conformed by PCR screen.Transgenic lines with the same growth and growth status were selected for q RT-PCR detection.Three high expression transgenic lines,OE7,OE28 and OE36,were used in further experimental studies.3.Stress tolerance analysis of Ms CAMTA2 transgenic tobacco（1）Phenotypic analysis of transgenic tobacco under salt,saline and dought stressPhenotypic observation of Ms CAMTA2 overexpression transgenic tobacco under stress showed that the transgenic tobacco presented less chlorosis and less wilt than that of wild type tobacco under all stress condition,especially under drought stress.Transgenic lines exhibited stronger recovery ability than that of wild type tobacco under drought stress.（2）Physiological analysis of transgenic tobacco under salt,saline and dought stressChlorophyll content,osmoregulation substance（proline,soluble protein and soluble sugar）,antioxidant enzyme activity（POD,SOD and CAT）and MDA content were measurement under stress,transgenic lines presented higher data in presented physiological assay but lower MDA content than that of wild type.In addition,the content of superoxide anion（O₂^-）and hydrogen peroxide（H₂O₂）in transgenic lines were lower than that in wild type tobacco,and DAB and NBT staining conformed that Ms CAMTA2 expressed transgenic lines accumulated lower ROS under abiotic stress than that of wild type tobacco.Otherwise,vertical plate root length assay with ABA added showed that transgenic tobacco perform more sensitivity to ABA treatment than wild type,and indicated that the stress response mechanism regulated by Ms CAMTA2gene may share crosstalk with ABA pathway.These results showed that the overexpression of Ms CAMTA2 gene in tobacco enhanced plant tolerance to salt,saline and drought stress.