Cloning Of Three Candidate Ginseng Genes And Expression Of Na ~ + / H ~ + Antiporter In Vacuolar Membrane

Reference gene is often used as the detection of gene expression levels because of its relative stability in various tissues and cells.Now, the most common reference genes include Actin, GAPDH,18s rRNA and EF1α,etc. In recent years, studies have found that these common reference genes have large different expression in different cell types and tissues, which would seriously affect the detection of gene expression analysis; therefore, it is important to select the appropriate internal reference genes.There reference genes were cloned from Euphorbia lathyris by RT-PCR and RACE and filtered out the most suitable one by FQ-PCR as internal reference to analysis the tonoplast Na+/H+ antiporter gene’s expression. It can be used to lay the basis of preliminary work to further improve plant salt tolerance mechanisms, the development and use of high-quality salt-tolerant gene resources. The main experiments are as follows:1 Three reference genes, including Actin、EF1α and α-tubulin, were cloned by method of RT-PCR and RACE and analyzed their homology, respectively.The result showed that all the amino acid sequence homology was more than 95%, indicating that these three genes are highly conserved genes.2 The quantitative PCR technology was used to select the most suitable reference gene from the three candidate reference genes in Euphorbia lathyris.The studies showed that the Actin gene has the highest stability and can be used as a reference gene in the study of EINHX gene’s expression.3 We isolated the vacuolar Na+/H+ antiporter cDNA fragment by RT-PCR and RACE technologys. The fragment contained 1334bp nucleotide acids, encoding a protein of 447 amino acids. Comoared with other plants, the homology of nucleotide sequence is above 75%. It showed that the gene we obtained is tonoplast Na+/H+ antiporter protein gene and its encoded protein is responsible for Na+ compartmentation to the vacuole.4 We used Actin as a reference gene to analyze the expression of Euphorbia lathyris NHX protein under the different tissues and different times NaCl treatment through fluorescent quantitative PCR. The experiment indicated the EINHX may be involved in the plant’salt tolerance mechanism,which provide the basis for the development of high quality salt-tolerant germplasm resources.