| Lycoris is a perennial herb belonging to Amaryllidaceae. It has high ornamental values due to its colorful flower and beautiful gesture. Lycoris also has the traits of strong adaptability, few diseases and insects, strong resistance, and so on. In this research, the physiological responses to drought stress of four species in Lycoris Herb.(Lycoris radiata, Lycoris aurea, Lycoris sprengeri and Lycoris chinensis) was studied, and drought resistance was also compared; The effect of three kinds of allogenic compounds on Lycoris radiata under drought stress was examined; Meanwhile, three genes related to drought stress(LrCMO, LrP5CS and LrRab) were cloned and their expression patterns were studied; By using agrobacterium-mediated transformation,19 independent transgenic rice plants overexpressing LrRab gene were obtained. The main results were listed as follows:1. Under drought stress, we measured the dynamic changes of water status, osmotic regulation substance, membrane system, protective enzyme activity and photosynthetic physiology, the results showed that with the extending of treatment time, root activity dropped sharply among the four species; leaf relative water content had different degree of decline; the trends of proline, soluble sugar and soluble protein contents were remained rise or firstly rised, and then declined; relative membrane permeability and malondialdehyde (MDA) content rised rapidly; the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were firstly increased, and then declined; chlorophylls contents declined all the time, while carotenoid contents were firstly increased, and then declined; photosynthetic ability declined in the all four species, and Lycoris chinensis had the greatest reduction. The drought resistance of four Lycoris species was evaluated by the method of fuzzy membership function and principal component analysis. Both results investigated by two methods showed that the drought resistance order of the four species was Lycoris radiata> Lycoris sprengeri> Lycoris aurea> Lycoris chinensis.2. Lycoris radiata leaf were pretreated with different concentration of CaCl2 solution, salicylic acid solution and sodium nitroprusside solution (0.1,0.5,2.5 and10 mmol/L) respectively for detecting the physiological responses under drought stress within 10 days. reactive oxygen species and its eliminating system were investigated. Comprehensively comparation of osmotic regulation substance, membrane system and protective enzyme activity suggested that 10 mmol/L CaCl2,2 mmol/L salicylic acid or 0.5 mmol/L sodium nitroprusside solution were the suitable pretreatment to enhance the drought resistance of Lycoris radiata.3. According to the amino acid sequence conserved region of CMO, P5CS and Rab genes in GenBank, the degenerate primer was designed. We obtained the full-length cDNA of the three genes from Lycoris radiata through degenerate primer PCR and RACE methods. They were named LrCMO, LrP5CS and LrRab respectively. qRT-PCR analysis showed LrCMO, LrP5CS and LrRab were expressed throughout the root, bulb and leaf of Lycoris radiata. The expression of LrCMO and LrP5CS were highest in bulb, while LrRab was highest in root. The expression pattern of three genes were first up-regulated and then down-regulated during drought stress. The expression fastigium of LrCMO and LrRab were at the 12 h, while LrP5CS was at the 6 h. We also constructed the eukaryotic expression vectors pCAMBIA1302-LrCMO and pPZP2Ha3-LrRab. Through the Agrobacterium-mediated transformation, we transfered recombinant plasmid pPZP2Ha3-LrRab into rice variety "Wu yun jing 7", and 19 independent transgenic plants were obtained, PCR detection results indicated all of them were positive. |
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