Functional Analysis Of Apple Plasma Membrane Intrinsic Protein Genes MdPIP1;1 And MdPIP1;2 Under Drought And Salt Stress

As an important membrane channel protein in plants,aquaporins(AQPs)form pores on the membrane to transport water and regulate the entry and exit of water on both sides of the cell membrane.Plasma membrane intrinsic protein(PIPs),as a subfamily of aquaporins,is a typical highly efficient water selective channel protein,which plays an important role in maintaining water balance in plants.In this paper,the full-length sequences of Md PIP1;1 and Md PIP1;2 genes were cloned from Malus domestica.cv.Qinguan and located in the plasma membrane.Transcription level of Mh PIP1;1 and Mh PIP1;2 genes were observed in leaf and root.To better understand the role of Md PIP1;1 and Md PIP1;2 genes in plant drought and salt stress tolerance,the transgenic 'Orin' apple callus and transgenic Arabidopsis thaliana overexpressing Md PIP1;1 and Md PIP1;2 genes was obtained.The main results are as follows:1.The full-length coding region sequence of Md PIP1;1 and Md PIP1;2 is 870 bp,which encodes 289 amino acids.Subcellular localization experiments showed that Md PIP1;1 and Md PIP1;2 proteins were located in the plasma membrane.The transcription level analysis of Mh PIP1;1 and Mh PIP1;2 genes showed that Mh PIP1;1 and Mh PIP1;2 genes were expressed in the top leaves,middle leaves,lower leaves and roots of apple,and the expression level of Mh PIP1;1 and Mh PIP1;2 genes in roots was higher than that in leaves.2.The transgenic 'Orin' apple callus and transgenic Arabidopsis lines overexpressing Md PIP1;1 was obtained.For Arabidopsis,under drought stress,there was no significant difference in relative electrical conductivity(REC),malondialdehyde content(MDA)and antioxidant enzyme activity between wild type and Md PIP1;1 overexpressed transgenic Arabidopsis thaliana,except that the survival rate of wild type was higher than that of overexpressed Md PIP1;1 transgenic Arabidopsis thaliana.Under salt 0.3 M Na Cl stress,there was no significant difference in survival rate,leaf relative electrical conductivity,MDA content and antioxidant enzyme activity between wild type and Md PIP1;1 overexpressed transgenic Arabidopsis thaliana.For apple callus,under the treatment of 0.06 M Na Cl and 0.08 M Na Cl,whether under normal culture conditions or under stress conditions,the phenotype and fresh weight of 'Orin' apple with Md PIP1;1 overexpression were significantly lower than those of wild type.In conclusion,the overexpression of Md PIP1;1 gene in Arabidopsis thaliana and 'Orin' apple callus did not change the drought tolerance of transgenic materials,but it seems to have a tendency to reduce the salt tolerance of transgenic materials.3.The transgenic 'Orin' apple callus and transgenic Arabidopsis lines overexpressing Md PIP1;2 was obtained.For Arabidopsis,under drought stress,compared with the wild type,the survival rate and antioxidant enzyme activity of transgenic Arabidopsis thaliana with overexpressing Md PIP1;2 were significantly higher than those of the wild type,so it could scavenge reactive oxygen free radicals more effectively to alleviate the plant damage caused by stress.The REC and MDA content of transgenic Arabidopsis thaliana leaves overexpressing Md PIP1;2 was significantly lower than those of wild-type,indicating that the degree of membrane damage in leaves was lower.Under salt 0.3 M Na Cl stress,compared with wild type,the survival rate and antioxidant enzyme activity of transgenic Arabidopsis thaliana with overexpressing Md PIP1;2 were significantly higher than those of wild type,and the relative electrical conductivity and MDA content of transgenic Arabidopsis thaliana with overexpressing Md PIP1;2 were significantly lower than those of wild type.For apple callus,under the treatment of 0.06 M Na Cl and 0.08 M Na Cl,compared with the wild type,the callus growth of 'Orin' apple with Md PIP1;2 overexpressed was faster,and the fresh weight was significantly higher than that of the wild type.As above,it's showed that overexpressing Md PIP1;2 gene in Arabidopsis thaliana and 'Orin' apple callus could improve the drought and salt tolerance of transgenic materials.