Cloning,Expression Analysis Of CsNHX6 Gene In Camellia Sinensis And Functional Identification Under Salt Stress

Tea plant[Camellia sinensis(L.)O.Kuntze]was originated in China,which has a long history of planting in our country,and the tea beverage made from its leaves is loved by people all over the world.However,affected by soil salinization,the soil environment suitable for the growth of tea trees is destroyed,which limits the increase in yield and quality of tea plant.In order to clarify the molecular basis and regulatory mechanism of tea plant response to salt stress,and also further provide theoretical basis on the molecular level for the identification and selection of tea plant salt-tolerant varieties.The main results are as follows:1.The Na~+/H~+antiporter gene CsNHX6 was isolated from the tea plant variety'longjing longye'.Bioinformatics analysis showed that the CsNHX6 gene contained a 1587 bp ORF,encoded 528 amino acids.Protein prediction analysis showed that the CsNHX6 protein contains 11 transmembrane domains,mainly located in the Golgi apparatus.Phylogenetic analysis showed that CsNHX6 had higher homology with VvNHX6 in grape and PtNHX6 in poplar,and that the polyclonal branch belonged to the NHX class-II subfamily,while had lower homology with AtNHX1,AtNHX2 and other class-I subfamily members.The results of qRT-PCR showed that the expression of CsNHX6 gene presented certain tissue specificity,and the expression was highest in the flower organ and lowest in the stem..Under the treatment of 200 mM Na~+and 120 mM K~+,the expression level of CsNHX6 gene was significantly induced,indicating that it was involved in the salt stress response process.At the same time,CsNHX6 gene was also induced by ABA treatment,and its expression level was significantly increased in a short period of time,speculated that it was involved in salt stress response and was dependent on ABA signal pathway regulation.In addition,CsNHX6 gene expression was inhibited under cold and heat stress,but the expression level did not change significantly by drought stress.2.Yeast functional complementation experiments showed that heterologous expression of CsNHX6 gene significantly increased the tolerance of salt-sensitive mutant strains GX3and AXT3 to Na~+and K~+.After 30 mM Na~+treatment,found that compared with the controls,transgenic yeast GX3 and AXT3 cells accumulated more Na~+and K~+,suggested CsNHX6 may also has the activity of Na~+and K~+transport.Further studies have shown that the transport activity of CsNHX6 may be affected by the H~+concentration.In addition,mutations in the key amino acid positions of CsNHX6 confirmed that D164,D193 and E188amino acids were essential for the Na~+/K~+transport activity of CsNHX6 protein,while the deletion of E320 amino acids had no effect on its transport activity.3.The overexpression vector pCAMBIA3302Y-CsNHX6 was constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation,and 5transgenic lines were obtained.Quantitative results showed that CsNHX6 was highly expressed in OE-1,OE-2 and OE-3 lines.After further screening to obtain homozygotes,the salt-tolerant function analysis of 3 high-expression transgenic lines was performed.The results showed that overexpression of CsNHX6 gene can significantly increase the germination rate of transgenic Arabidopsis seeds under salt stress.The above results indicate that CsNHX6 gene is involved in the salt stress response process and plays an important role in improving plant salt tolerance.