Analysis Of The Salt-tolerant Genes Expression Profile And Identification Of The Salt-tolerant Genes

Soybean is an important cash crop and its productivity is significantly hampered by salt stress. Development of salt tolerant cultivars of interest is very necessary. Despite wealth of information generated on salt tolerance mechanism,its basics still remain elusive. To understand the molecular basis of soybean responses to salt stress better and clone several critical salt stress response genes in soybean. SSH libraries (SSH1 and SSH2) were constructed for the root tissue of two cultivated soybean genotypes a salt-tolerant cultivar and a salt-sensitive cultivar, to compare salt treatment and non-salt treatment plants. Some modern biotechnologies were used to compare and analyze between the two libraries, several ESTs got might be related highly to salt tolerance, and their expression under the salt treatment and no-salt treatment were investigated and analyzed. Two ESTs were selected and their full lengths were cloned. They were translated into the soybean roots and Lotus corniculatus plants to study the affects on the tolerance. The results were described as followed:SSH libraries (SSH1 and SSH2) were constructed for the root tissue of two cultivated soybean genotypes: Wenfeng7,a salt-tolerant cultivar,and Union, a salt-sensitive cultivar, to compare salt treatment and non-salt treatment plants. The titre of the SSH1 is 10000 by transformation and counting, the same number in SSH2. The length of the insert fragments varied from 200 to 500bp.Dot blot analysis show that there were 262 differentially expressed cDNA clones in SSH1 and 121 differentially expressed cDNA clones in SSH2. All the differentially expressed clones were sequenced. In total, 377 high quality EST sequences were got, 260 from SSH1 and 117 from SSH2, and these ESTs have been loaded into dbEST with accession numbers GT917016-GT917393 and GU385812. 61 genes from SSH1 could be assigned putative function on the basis of the sequence similarity to the genes or proteins of known function in the GenBank, and 49 genes from SSH2.Comparing and analyzing between the two SSH libraries show that the putative function of 32 genes from SSH1 was similar and same with some genes from SSH2. In SSH1, there were eleven pairs of genes whose sequences were similar or same. These sequences may be not the salt tolerance-related genes, remove the genes from SSH1,the remains might contain more genes related to salt tolerance. The result of this study showed that the novel test way could low the background of SSH and enrich the salt-tolerant genes to some extent.20genes were selected to analyze their expression under salt treatment and no-salt treatment from the remains in SSH1, of these twelve unigenes with matches in non-redundant protein database and eight with no match. Full-length or 3'and 5'sequences of seven genes were forecasted by electronic elongation. Two genes which were involved in plasma membrane intrinsic protein and ANI-zinc finger family protein respectively might be the salt tolerance-related genes. The primers were designed based on the forecast sequences and they were cloned by RT-PCR. Their sense and anti-sense expression vectors were constructed, and the two genes were translated into the soybean hairy roots and Lotus corniculatus plants to study their affects on the salt tolerance.