Isolation And Target Of Salt-responsive Genes In Salt-tolerant Somatic Hybrid Line Of Wheat As Well As Study On Their Molecular Mechanism Of Salt Tolerance

To respond to the adverse environmental stresses, a complicated and coordinative regulation net differing in time and space is developed in plant. Salt stress condition may activate multiple signaling pathways, and activate or inhibit downstream effect genes, including genes related with ion transport and reestablishing homeostasis, genes that participate in the biosynthesis of osmoprotectants, genes related with water-deficit stress and genes related to scavenging ROS or preventing them from damage of cellular structures, etc. All of these genes make full functions coordinately to keep plant growing and developing regularly.The salt-tolerant traits could be successfully transferred from alien species to common wheat by asymmetric somatic hybridization. In our laboratory, the asymmetric somatic hybrydization between common wheat Jinan 177 (Triticum aestivum L.) and intergeneric grass (Agropyron elongatum Host Nevski) had been carried out and the hybrid wheat progeny lines were first obtained in the world. Through selection in many years and test for the salt tolerance of the wheat progeny both in the laboratory and in the field, a new salt-tolerant wheat variety Shanrong No.3 has been delivered, which can produce a high yield in the soil with 0.3%~0.5% salinity .In this thesis, our goal is to study the salt-tolerance mechanism of the somatic hybrid Shanrong No.3 and investigate the genetic basis of its salt-tolerance. By using both mRNA differential display technique and 5' RACE method, two full-length cDNA related to salt stress were cloned from Shanrong No.3 and their expression characters were analyzed. In addition, SSR markers were employed to target the salt tolerance genes of Shanrong No.3, and a major gene controlled salt tolerance has been located on the wheat chromosome.The main research contents and results achieved in this work were summarized as follows.1. Isolation and Analysis of cDNA Fragments Responded to Salt-stress From wheatsomatic hybrid Shanrong No.3 by mRNA Differential DisplayIn this research work, the different expression in salt-stressed and unstressed sensitive parent wheat line Jinan 177 and salt-tolerant wheat variety Shanrong No.3 from asymmetric somatic hybridization between wheat Jinan 177 and Agropyron elongatum were analyzed by the silver staining mRNA differential display method. Total 106 salt-induced cDNA fragments, expressing differentially in Jinan177 and Shanrong No.3, were isolated. Among them, 87 derived from roots, and only 19 from leaf. The results showed that there are more differential genes expressed in roots than in leaves when treated with 200mmol/L NaCl for 6-72hrs.Out of 27 positive cDNA fragments screened by reverse northern blotting, 10 cDNA fragments were sequenced and analyzed. The deduced amino acid of wrsi-1 showed homologous to peroxidase from Asparagus officinalis and Gossypium hirsutum. The deduced 54 amino acid sequence of wrsi-2 exhibited 51% identity to that of root hair defective gene. The fragment wrsi-5 indicated homology to ESTs from many abiotic stresses. As the key point, all three fragments mentioned above would be studied. In addition, a lot of genes, in relation to protein synthesis and metabolism and with cell detoxification and defensive (such as wrsi-3. 4 and wlis-1), were expressed under salt stress. And some genes, which were involved in salt-stress response (wrsi-6. 7. 8 and wlis-2), but their function were unknown, need to be verified next.Based on the analysis of the deduced amino acid sequence, it is inferred that wrsi-1 encodes peroxidase , which is a member of ClassIII group. The expression of wrsi-1 in the root of Shanrong No.3 induced by salt stress was further confirmed by Northern blotting, whereas the hybridization signal was not detected in Jinan 177. So, the wrsi-1 may be an early salt responding gene, and it is deduced that the up-regulated expression of this gene can play a major role in scavenging O2- and other toxin, as well as preventing the cellular structures from damage..2. Cloning of the Full-length cDNA of the Shanr...