Cloning And Functional Identification Of MdeSOS1 Gene In Magnolia Denudata

Magnolia denudata belongs to the deciduous tree of Magnolia plants,which has important ornamental value,and it is an excellent flower and tree species in early spring in China.In the process of cultivation and application,it is found that Magnolia plants are difficult to grow and development in some regions where soil salinization is serious.Our research group previous studies had found that M.denudata exhibited a certain degree of resistance under salinity environmental,and its relative conductivity,malondialdehyde,proline,soluble sugar and peroxidase were positively correlated with the concentration of sodium chloride.However,the molecular regulation mechanism of salt tolerance remained to be elucidated.In this study,we analyzed the tolerence mechanism of M.denudata to salt stress at the molecular level and the function of key salt resistance gene.Our results could provide reference for the study on improving the salt resistance of Magnolias.The main conclusions are as follows:(1)Ion content determination results showed that Na~+content in root,stem and leaf in M.denudata rose by 64.31%,1087.34%and 225.44%compared with 0 h,and potassium ion in the stem decreased most obviously,which reached 82.65%.The strong interception of Na~+by the stem ccould reduce the transport of sodium ions into the leaves and reduce the damage of the leaves.At the same time,the K~+was transported from the stems to the roots and leaves,which helps the plants to cope with salt stress.(2)The softwares of geNorm,NormFinder and BestKeeper were used to analyze and screen optimum reference genes from 13 candidate genes in different tissues when M.denudata exposed to salt stress.The results showed that GBP,UBQ and UBC could serve as stable reference genes for gene expression among different tissues,while 18S was the worst reference gene.The results will provide a reliable reference gene for the study of the expression of target gene in Magnolia under saline stress.(3)MdeSOS1 gene cloned by RT-PCR belongs to the traditional plasma membrane Na~+/H~+reverse transport protein family,and its coding sequence contains the Nhap region,which is responsible for ion transport and metabolism.Amino acid sequence comparison revealed that the identity between MdeSOS1 and AtSOS1 was 62.7%,and with the PeSOS1 was 68.3%.The phylogenetic tree showed MdeSOS1 was a single branch in the evolution process of SOS1s and located in the relatively original status of dicotyledons The MdeSOS1 protein was located in the plasma membrane and it played the role of transport ion on the membrane.Fluorescence quantitative analysis displayed that MdeSOS1 was preferentially expressed in stem and root,and the total expression in stem segment was higher than that in root and leaf.It is supposed that the high expression of MdeSOS1 in stem contributes to the regulation of sodium-potassium ion flow and determines the distribution of ions in different organs,so that plants could better adapt to salt stress.(4)MdeSOS1 was expressed under the control of 35S promoter and transferred into Arabidopsis sos1-1 mutant and wild-type genomes by Agrobacterium-mediated inflorescence infection transformation.The results demonstrated that the transgenic plants displayed better adaptability under salt stress compared with the control,which further verified the salt tolerance function of MdeSOS1 gene.