Functional Charcaterization Of The Aabiotic Stress And Identification Of Up-stream Regulation Of ThTIP From Tamarix Hispida

Aquaporins?AQPs?are integrated membrane proteins of a class of highly efficient and specific transporting water molecules.In the environment of salt stress,the mechanism of plant regulation of water in and out of cells is mainly mediated by AQPs.It can affect the permeability of cell membrane through various activity regulation methods,which plays an important role in maintaining cell osmotic balance.TIPs?tonoplast intrinsic proteins?are a subfamily of the plant AQP family,located in the vacuole membrane,and thus become the vacuolar membrane intrinsic protein.In order to deepen the understanding of AQPs function,improve the resistance to salt regulation mechanism of woody plants,this study intends to ThTIP under salt stress expression of time and space characteristics,ThTIP upstream regulatory factors and ThTIP of plant salt tolerance physiological regulatory system in three aspects:the research of tamarisk ThTIP gene in the process of salt stress response function.In this study,a aquaporin gene,named ThTIP,was identified from the data of Tamarix chinensis.The gene was 759 bp in length and encoded 252 amino acids.It encodes the protein relative molecular mass 26099.25Da,the theoretical isoelectric point?PI?is 4.6.Amino acid sequence analysis showed that ThTIP contained six transmembrane regions and two water channel protein-specific NPA?Asn-Pro-Ala?units.After constructing the plant overexpression vector of ThTIP,Arabidopsis thaliana was transformed and the transgenic Arabidopsis plants of T₃ generation were stained,The wild type and transgenic lines of Arabidopsis were treated under NaCl and Mannitol.The results showed that compared with WT plants,transgenic lines had higher germination rate,root length,fresh weight and growth.The two lines of the T₃ generation of ThTIP transformed plants displayed significantly improved tolerance to salt and drought.A suppression vector of pFGC5941-ThTIP was constructed.By using transient transformation method,three kinds of transgenic T.hispida plants were generated,including plants overexpressing ThTIP?transformed with pROK?-ThTIP?,RNAi-silencing ThTIP plants?transformed with pFGC5941-ThTIP?,and control plants?transformed with empty pROK??.DAB,NBT,and Evans Blue staining showed that compared with control plants and RNAi-silenced ThTIP plants,plants overexpressing ThTIP has lowest ROS content,and suffers lowest cell membrane damage.These results suggested that overexpression of ThTIP significantly improved abiotic stress tolerance of plants.Physiological indexes sush as water loss rate,chlorophyll content,proline content,H₂O₂ content,MDA content,POD and SOD activity were measured to analysis the stress tolerance in the three kinds of plants.The results showed that,the water loss rate,ROS and MDA content in transgenic plants overexpressing ThTIP were lowest,and the overexpression plants showed highly increased chlorophyll content,proline content,SOD and POD activity.These Indicated that overexpression of ThTIP in transgenic T.hispida plants increased the reactive oxygen species?ROS?scavenging capability,decreased cell membrane damage,and then improved the plants'tolerance to stress.The deletion fragments of the 5'end of the ThTIP promoter were obtained by a set of 5'deletion method,named N1?-1^-1731 bp?,N2?-1^-1299 bp?,N3?-1⁸67bp?,N4?-1^-432bp?and N5?-1¹81 bp?.The five fragments were replaced with the CaMV 35S promoter on the expression vector pCAMBIA1301,and the plant expression was constructed with GUS gene Vector,and transformed Arabidopsis thaliana.The results of GUS staining showed that N1-N4 fragment could be stained blue,but the fragment of N5 could not be stained,that means the 251 bp?-181^-432bp?sequence of N4-N5 was the core region of ThTIP promoter segment.There are three cis-acting elements in the core segment by Plant CARE online analysis,which may be combined with the upstream regulatory factors and then control the TIP promoter,so we mutatied of the core fragment promoter one by one and then built to pCAMBIA1301-35S::GUS vector.The transformation of Arabidopsis thaliana under salt stress showed that the ABRE1 and ABRE2 element might be an important response on the ThTIP promoter under salt stress.The binding of ABRE and ThABFs was identified by yeast single hybridization.First,the effect vector of pHIS2-ABRE and the effect vector pGADT7-Rec2-ThABFs was constructed.The ABF family gene of ABRE was screened by yeast single hybridization.The results showed that some ABRE/ABF family genes ThABF1,ThABF2,ThABF7,ThABF8 recognizes and binds to the ABRE element of the ThTIP promoter to regulate ThTIP gene expression.In order to verify the results of yeast single hybridization,the ABRE element was reconstructed on a pCAMBIA1301-46bp reporter vector with 35bp small promoter substitutions for 35S,and pROK?-ThABF1,pROK?-ThABF2,pROK?-ThABF7,pROK?-ThABF8 was constructed as the expression vector of GUS gene,and then transiently infect tobacco with reporter vector and effect vector by Agrobacterium tumefaciens-mediated method.The expression of GUS was detected,the results showed that ThABF1,ThABF2,ThABF7,ThABF8 and ABRE were differentially bound with ABRE elements,which confirm that ThABF1,ThABF2,ThABF7and ThABF8 transcription factors could interact with ABRE elements.