Functional Analysis Of PtMAPKK6 Gene In Response To Abiotic Stress

As one of the halophytes,Puccinellia tenuiflora has strong salt tolerance and is considered as a good material for study on molecular mechanism of stress resistance in plants and isolation of stress resistant genes.In our previous study,PtMAPKK6 gene was isolated from Puccinellia tenuiflora and found to be widely involved in response to low temperature,drought and salt stresses in Puccinellia tenuiflora root.On this base,the PtMAPKK6 gene was transformed into pbs2?PBS2 encoding yeast mitogen-activated protein kinase kinase,MAPKK?deficient yeast strain,and MAPKK function-complementary yeast strain was obtained.The growth potential of MAPKK function-complementary yeast strain under stressed conditions was analyzed,and the wild type yest strain and pbs2 mutant strain were used as control.The introduction of PtMAPKK6 gene in pbs2 mutant yeast improved the resistance of yeast to osmotic stress,low temperature and NaCl stress,which indicated that PtMAPKK6 gene might participate in signal transduction in pbs2 deficient yeast in response to osmotic,low temperature and NaCl stress.In order to further study the function of PtMAPKK6,the plant overexpression vector of PtMAPKK6 gene and the RNAi expression vector of AtMAPKK6 gene were constructed and tranformed into wild-type Arabidopsis thaliana by Agrobacterium mediated floral dip method,respectively,and the empty vector was used as control.After the hygromycin and PCR selection,T2 transgenic plants were obtained.By the real-time fluorescence quantitative PCR technique,the expression of target gene in T2transgenic plant was analyzed.The PtMAPKK6 transgenic line with middle expression level and the AtMAPKK6 transgenic line with middle suppressed expression level were selected and treated with PEG and Na₂CO₃,and the empty vector transformed plant and wild-type Arabidopsis plant were used as control.The enzyme activity of POD,SOD and CAT in the leaf of the transgenic plant under the stressed condition was assayed.The results indicated that the enzyme activity of POD,SOD and CAT in PtMAPKK6overexpressed plants was higher significantly than the empty vector transformed plants and wild-type plant.The enzyme activity in the transgenic plant with suppressed expression of AtMAPKK6 was obviously lower than wild-type and the empty vector transformation plant.PtMAPKK6 gene may be involved in the response of Arabidopsis thaliana to adverse stress through effect on antioxidant enzyme activity.