Functional Study Of HgNHX1 Gene And HgNHX1 Promoter In Halogeton Glomeratus

Soil salinty is one of the major abiotic stresses in china,it can affect the normal physiological activities and crop yields.Salt stress can lead to high Na~+ concentration in the soil,resulting in destruction of plant ion homeostasis and osmotic stress,which have an inhibitory effect on seed germination,growth and photosynthesis.Vacuolar Na~+/H~+ antiporter play an important role in the ion balance,salt tolerance and the whole development process of plants.Halogeton glomeratus(H.glomeratus)is one of an annual dicotyledonous plants belong to Chenopodiaceae.They mainly distributed in Gansu,Qinghai,Xinjiang and Tibet,mostly existed in grassland,saline-alkali land of desert and semi-desert areas.H.glomeratus is one kind of halophytes,it can grow normally in highly saline environment,which leaves and stems are highly carnified.It is a good material to study the salt tolerance gene and the mechanism of salt tolerance,it has important application value for improving crop resistance and cultivating saline alkali tolerant crops.The main mechanism of salt tolerance is to separate the toxic ions(Na~+,Cl-)in the vacuole,So studying the H.glomeratus regulation mechanisms of salt tolerance using molecular methods,seeking a large number of genes which have potential applications in drought resistance and salt tolerance breeding will have a practical sense in enriching salt-tolerant gene pool and cultivating salt-tolerant crops.The commonly used promoter is the 35 S promoter which is a kind of constitutive promoter,it can be efficiently expressed in tissues and organs.However,the expression of exogenous gene will break the balance of metabolism,which will affect the growth of plants.Therefore,in order to reduce the adverse effects,cloning of a new promoter with tissue specificity plays an important role in genetic engineering.Vacuolar Na~+/H~+ antiporter gene HgNHX1 and the promoter(p HgNHX1)in H.glomeratus was isolated and tested the function in this study.The main results of this experiment are as follows:1.The upstream of 5' flanking region of HgNHX1 gene was cloned,and used the Plant CARE and PLACE to analyse isolated sequence.The results showed that besides the TATA-box and CAAT-box,a number of potential cis-elements and transcription binding motifs which are related to stress responses were found,such as salt,dehydration,cold and wounding responsive elements.Other potential cis-elements which are responsive to phytohormones were also found in the sequence.Analysis of the sequence indicated that it has general characteristics of typical promoter.2.Constructed a plant expression vector: p BI-p HgNHX1.Imported into Arabidopsis and tobacco successfully,the function was verified by GUS histochemical staining.Results showed that: four promoters of different length can drive the expression of GUS gene in tobacco leaves and Arabidopsis thaliana,it indicated that the promoter had promoter activity and ?1 already contains the core element,but the smallest fragment was lighter than the other three.3.GUS histochemical staining was performed on tobacco and Arabidopsis thaliana which had been successfully transformed ?4 promoter.The results showed that GUS gene can be expressed in tobacco leaves and stems and also can be expressed in Arabidopsis thaliana's different growth stages and different tissues,so we can indicated that the promoter has constitutive promoter activity.4.In this experiment,we used the constructed expression vector p CAMBIA1301-HgNHX1 to transform Arabidopsis thaliana,then the function of salt and drought resistance was tested.The results showed that the gene was over expressed in transgenic Arabidopsis leaves under drought and salt stress,but there was no significant difference in the phenotype of transgenic Arabidopsis under stress compared with the wild type Arabidopsis,and further through the determination of physiological index RWC,proline content,chlorophyll content and MDA,we found that there were no significant differences between the transgenic plants leaf and WT plants under salt stress.The results showed that the over expression of HgNHX1 gene did not improve the drought resistance and salt tolerance in Arabidopsis.