Isolation And Characterization Of Responsive Genes Involved In Adversity Stress In Leymus Multicaulis

The soil salification is a serious problem for agriculture and environment. Especially now with the development of industry, the situation of soil salification becomes more and more serious, and the quality of water is continuous worsened. Research on the mechanism of salt tolerance especially in the important crops, such as wheat, is becoming more urgent than ever before. Barley Yellow Dwarf Virus often leads to a serious loss in the yield of wheat. But at present, we have not find excellent genes resistant to BYDV in wheat, so transferring the excellent genes from relatives to cultivars of wheat will be a good method. The leymus multicaulis has many excellent traits, such as immune or highly resistant to BYDV, and highly tolerant to salt and aphid, which are controlled by some unknown genes. So it is very important to isolate and clone these genes in leymus multicaulis for utilizing in breeding.In this research, DDRT-PCR technique was used to study these genes and two common wheat- leymus multicaulis Disomic addition lines were included. One of them is Line24 which is tolerant to BYDV and aphid when inoculated with aphid which take GAV strains. The other is Line 14 which is tolerant to salt when treated with 1% NaCl solution.The result showed that fourteen fragments which is only expressed in Disomic addition line-Line 14 and twelve fragments only expressed in Disomic addition Iine-Line24 were obtained. These fragments were cloned in E.coli DH5 a competent cell after having been ligated with the pUcm Vector . By sequencing and then querying EST database of GenBank, we can find that some fragments have high homology to known genes. Six fragments that showed low homology to known genes were tested with northern blotting. The results showed that NB14 and NY 13 had positive hybridization signals, but no signal in the parent-common wheat, and NB14 fragment only had positive signal hybridized with Line24. Therefore, we can not deduced it is a responsive gene at present. Much work still needed to confirm it. NY13 fragment had positive signals hybridized with Line 14 and leymus multicaulis, which proved that it was a responsivegene in salt stress. Furthermore, we tested NY 13 with southern blotting, and also found positive signal in the leymus muhicaulis. This segment may be correlated with the addition chromosome and has the character of salt-resistance .The blot fragments in lane of leymus multicaulis probably included the new responsive genes to salt stress. Some fragments were reclaimed and cloned in E.coli DH5 a competent cell with the TAC vector. Such clones may have the full length genes. Then we can select salt-stressed relative genes from these TAC clones and it can be further applied to genetic engineering in crop breeding in the future.