Cloning, Sequence Analysis And Genetic Transformation Of The Salt-tolerant Related Genes From Thellungiella Halophila

Salinity soil is a worldwide problem threatening crop production.Salt stress is the major factor that limits and decreases the output of crops in many parts of the world, particularly for the irrigated land.High salinity of soil causes salina and limits soil utilization.Na~+ in salinity soil is the major factor of inhibiting plant growth.But for most plants,Na~+ is the primary cause of ion-specific damage.Therefore,clone and analysis Na~+-transport related genes are valuable to study salt-tolerant mechanism.Many,too many,species have been used to examine salinity response physiology, but the names associated with genetic or molecular genetic studies in salinity stress research are few.The recent discovery of the halophytic plant species,Thellungiella halophila,which is native to the seashore saline soils,meets all the criteria for being a genetic model system.Thellungiella halophila which is a relative of the Arabidopsis has small size,short life cycle,self-pollination,and high seed number,and favorable genetic traits such as self-fertilization,a small genome,efficient transformation,and mutagenesis. So,Thellungiella halophila becomes the prospective salinity tolerance model.The major purpose of our experiment is using Thellungiella halophila as the research material, studying the function of the salt related gene of ThNHX1 and ThHKT1.1.A cDNA fragment was amplified from Thellungiella halophila using primers designed based on the Arabidopsis thaliana AtNHX1 gene sequence in RT-PCR.5'- and 3'-RACE methods were then used to obtain a full length cDNA.The cDNA sequence was 2045bp in length including an open read frame of 1638bp,encoding a predicted polypeptide of 545 amino acids.The detailed sequence analysis showed that the gene shared the highest homologous with AtNHX1.Thus,the cDNA encodes a putative Na~+/H~+ antiporter(ThNHX1).In addition,the gene structure of ThNHX1 based on intron and exon analysis had high similarity to that of AtHNX1.The phylogenetic analysis of various Na~+/H~+ antiporters indicated that ThNHX1 is grouped with tonoplast Na~+/H~+ antiporters,and distinguished from plasma membrane Na~+/H~+ antiporters.2.Basing on routine molecular cloning techniques and using the PGEM-T vector, the coding region of ThNHX1 was inserted into the selectable marker-removable expression vector PX6-GFP.The recombinant vector was introduced into Agrobacterium tumefaciens LBA4404 with freeze-thaw method.Tobacco was transferred by leaf disk, transformants of tobacco that screened by kanamycin medium were obtained.3.Using Thellungiella halophila as the research material,a cDNA fragment was amplified from Thellungiella halophila using primers designed based on the Arabidopsis thaliana AtHKT1 gene sequence in RT-PCR.RACE methods were then used to obtain a full length cDNA.The cDNA sequence was 1817bp in length including an open read frame of 1503bp,encoding a predicted polypeptide of 500 amino acids.And PI is 8.82.The detailed sequence analysis showed that the gene shared the 82.8%similarity with AtHKT1.