| Due to persistent shrinkening of agricultural area, it has increasing demand in development of the saline-alkaline soil. One of the strategies for crop breeding is to increasing plant tolerant capability to saline-alkaline stress through genetic improvement. Leymus chinensis (Trin.) Tzvel., a xerophilous grass, is adapted to saline-alkaline soil. It must have developed molecular and physiological mechanisms to adapt itself under such stress conditions. Therefore. Leymus chinensis is considered a material for study the molecular mechanisms of saline-alkaline tolerance.In this study, three genes encoding chitinase. thioredoxin peroxidase and vacuolar H+-ATPase c subunit were cloned from Leymus chinensis EST library by RACE and registed on the NCBI (GQ397277. GQ397275 and GQ397276) named LcChi2. LcTpxll and LVAPL. respectively. The nucleic acid and protein sequences of these genes were analysis by bioinformatics methods, and subcellular localization of these three genes were tested by confocal laser microscopy. LcTpxlI was expressed and purified from E. coli. and the activity of the purified protein was analyzed. The results showed that LcTpxlI could not only reduce H2O2. but also protected of the plasmid antioxidant. Semi-quantitative RT-PCR was used to analyze the expression of the three genes under saline-alkaline stress. The results showed that the expressions of the three genes were up-regulated under saline-alkaline stress, indicating that these genes were responds to Leymus chinensis saline-alkaline tolerance. To further demonstrate the function of the three genes, the three genes were transformed into yeast. The transformed yeast grows better than the control under saline-alkaline stress, indicating that the three genes could increase the saline-alkaline tolerance to transgenic yeast. Finally. LVAP1 and LcChi2 were transformed into tobacco. The results showed that the transgenic tobacco grows better than the wild type under saline-alkaline stress, indicating that the two genes could improve the ability of transgenic plants against saline-alkaline stress. Meanwhile, the disease resistance of the LcChi2-transformed tobacco was improved, indicating that LcChi2 conferred the resistance of bacterial and fungal pathogens to the transgenic tobacco.Taken together, we successfully obtained three genes which were associated with saline-alkaline tolerance from Leymus chinensis EST library by RACE and registed on the NCBI. The functions of these genes were verified by transformed yeast and transgenic tobacco. These genes may be the interesting candidate genes to be used in breeding to improve saline-alkaline stress in crops and reform the saline-alkaline soil. |
