Evaluation And Gene Analysis Of Salt Tolerance On Wild Soybean Germplasm From Henan And Shanxi Provinces

Soil salination is one of the major abiotic-stress that influences agricultural production and environment. With the growing of world's population and the decreasing of field, how to utilize salinized-soil becomes more and more urgent. Screening the salt-tolerant germplasm resources, cloning the genes related to salt-tolerance, and breeding new salt tolerant varieties are the most economic and efficiency strategy to improve and utilize salinized-soil.The Glycine max (G. max) is the domestic species origin from Glycine soja (G. soja). G. soja is the plentiful resources for the G. max improvement. For screening the high salt tolence germplasm of G. soja, 284 wild soybean germplasm that introduced from Henan and Shanxi province have been identified and evaluated at three different stages of germination, seedling and whole period of growth. Some SNPs related to the salt tolerance have been detected using PCR-SSCP and salt relative gene has been cloned using sequence blast analysis. The function of gene has been identified using Prokaryotic expression system and Arabidopsis thaliana (A. thaliana) transformation systems.The study has got following results:1. Evaluation of salt-tolerant capacity at three stages of germination, seedling and whole period of growth in wild soybean germplasm284 wild soybean germplasm that introduced from Henan and Shanxi province have been identified and evaluated at three different stages of germination, seedling and whole period of growth. At stage of germination, 8 accessions are grade 1, 31 are grade 2, 68 are grade 3, 98 are grade 4 and 79 are grade 5. Meanwile, at stage of seedling, 19 are grade 1, 20 are grade 2, 26 are grade 3, 80 are grade 4 and 127 are grade 5. ZYD03676,ZYD03572,ZYD03653,ZYD03666 and ZYD03672 have high tolerance on the farm identification.2. SNP analysis of genes related to salt-tolerance in wild soybean using the method of PCR-SSCPAccording to the cDNA sequences of 8 salt-tolerance relative genes in Genebank, some pairs of primers were designed and the size of PCR product is between 150bp to 300bp. After analysis of the wild soybean germplasm which grade is intermediate using PCR-SSCP technique, some different sites of EREBP1 were found in the population of ZYD03499 and ZYD03499 can be divided into two groups according to the type of bands.3. Cloning of EREBP1 gene in wild soybeanThe full-length fragment of EREBP1 gene has been tested and the size of it is the same to the cDNA in Genebank. So, there is no intron in this gene. After blasting two types of EREBP1, 7 different sites were found in type 2. Among them, 5 are synonymous mutation and 2 sites (248 and 536) have changed the amino acids. 4. Function analysis of EREBP1 gene in wild soybeanFusion expressing vector pET30a+ was constructed which contain EREBP1 gene, and expression of fusion protein was induced by IPTG. The expression products were analyzed by SDS-PAGE. A new band was induced and the expression quantity of EREBP1 fusion protein was the most after induced 4 hours. Transformed-gene vector for dicotyledonous plants pBI121 was constructed which contain EREBP1 gene. EREBP1 was transformed to Arabidopsis by Agrobacterium infiltration method. The transgenic plants were obtained according to Kana resistance.