Cloning And Expression Analysis Of The Genes Related To Salt Stress Tolerance In Leymus Multicaulis

Leymus multicaulis. is one of important forage grasses with a lot of excellent traits such as salt-tolerance, drought-tolerance, leanness-tolerance, BYDV (barley yellow dwarf virus)- resistance aphid-tolerance et al, Which is important for the further development of salt-tolerance crop breeding. In this research, we studied the isolation of total RNA from the leaves of Leymus multicaulis and the improvement of the DDRT-PCR system suitable for analysis of differential mRNAs in leaves and the cloning and function analysis of the cDNA segments related to salt tolerance. And we also cloned glutathione reductase gene and analyzed its expression under salt stress. The main results of experiment were showed as following:Total RNA from the leaves of Leymus multicaulis was isolated by using different methods: guanidine thiocyanate, SDS, CTAB and TRIZOL. The yield and purity of RNAs were analyzed by electrophoresis and spectrophotometer. The results showed that the RNA isolated by TRIZOL presented three clear bands—28S rRNA, 18S rRNA and 5S rRNA respectively, and its purity (A₂₆₀/A₂₈₀: 1.845, A₂₆₀/A₂₃₀: 2.091) is higher than other three methods although a lower yield, This indicated that the method of TRIZOL is one suitable for total RNA extraction from the leaves of Leymus multicaulis.A DDRT-PCR system was established by perfecting the conditions of Mg²⁺, annealing temperature, template concentration and Taq enzyme. 17 perfect differentially expressed cDNA segments were isolated by mRNA differential display. Reverse Northern analysis indicated that 11 of 17 segments were positive clones.Sequence analysis and homologous comparison of those cDNAs with the help of Genbank/BLAST revealed that: protein of cDNA No.2 has 42% similarity to UMP/CMP kinase gene; segment No.4 cDNA has 99% similarity to that of Sorghum bicolor under water stress, which encodes S9 protein and probably participated in protecting plants from environmental oxidative stress; protein of segment No.5and 10 have 35% similarity to...