Cloning And Functional Analysis Of AcTIP1;3 And AcTIP4;1 Encoding Tonoplast Intrinsic Protiens From Atriplex Canescens

Drought and salinity lead to reduction of plant growth,productivity and quality,however,most forages and crops are glycophytes,and their tolerance to salinity are limited.The halophytic species inhabiting in the drought and/or saline environments have enlvoved mutiple unique stress tolerant mechanisims,contain abundant stressresistant genes.Orginated from the northen America,fourwing saltbush(Atriplex canescens)is a typical secretohalophyte with excellent salt and drought tolerance since its salt baldder cells can sequestrate a massive amount of salt and finally secrete the salt after bladder cells rupturing.In this process,the water is also transported into salt bladder cells.However,the mechanism how does water entry into the salt bladder cells during salt sequestration remains unkonwn.The plant aquaporin tonoplast intrinsic protines are mainly located on the tonoplast membranes to mediate the transportation of water and other small molecules and play important roles in plants response to biotic and abiotic stresses.According to transcriptome sequencing,we previously found that the expression of two TIP genes of A.canescens were significantly up-regulated by Na Cl treatment,suggesting that they may be involved in water transportation of salt bladder cells of A.canescen.By sequence alignment,above two genes shared high similarity with homologous genes TIP1;3 and TIP4;1 from other plant species,respectively,and thus were named as AcTIP1;3 and AcTIP4;1.Therefore,in this study,the c DNAs of above two tonoplast intrinc protiens were cloned from A.canescen,their subcellular localization were analyzed and their water transport capacity was identified by heterologous expression in Saccharomyces cerevisiae and the oocytes of Xenopus Laevis.Finally,we transferred AcTIP1;3 gene into Arabidopsis thaliana to determine its role in drought tolerance of transgenic plants.The main results are as follows:1.The full length of c DNA encoding AcTIP1;3 and AcTIP4;1 were cloned.Both of them contain an open reading frame(ORF)of 747 bp,which encodes 248 amino acids.Transmembrane domain analysis shows that AcTIP1;3 has seven transmembrane helices,while AcTIP4;1 had six transmembrane helices,and both of them had two NPA domains,which are typical domains of the major intrinsic protines family.2.Yeast heterologous expression analysis showed that AcTIP1;3 and AcTIP4;1enhanced the osmotic tolerance of transgeneic yeast;The Xenopus oocytes heterologous expression analysis found that AcTIP1;3 and AcTIP4;1 caused oocytes swellen and brust,indicating both AcTIP1;3 and AcTIP4;1 exhibited excellent water transport ability.3.By the transient expression in tobacco leaf epidermal cells,we found that AcTIP1;3 and AcTIP4;1 mainly located in the vacuolar membrane.4.The transgenenic lines of A.thaliana overexpressing AcTIP1;3 were obtained.Under drought stress,overexpression of AcTIP1;3 in A.thaliana increased the biomass,reduced relative plasma membrane permeability and MDA content,and increased enzyme activity of SOD,POD and CAT of transgenic plants.The overexpression of AcTIP1;3 in A.thaliana improved the resistance of transgenic plants to drought stress.In summary,the tonoplast intrinc protiens AcTIP1;3 and AcTIP4;1 of A.canescens were involed in water transport,and all above results laid a theoretical foundation for water accumulation of salt bladder cells of A.canescens improved salt and drought tolerance of other plant through genetic methods.