| Zoysiagrass and bermudagrass are typical warm season turfgrass,mainly distributed in the south and north part of China’s Yangtze River.Low temperature injury is a disaster to warm season turfgrass in the northern area,it will not only lead the growth damage of turfgrass,even cause plant death. The study on the damage mechanism of warm season turfgrass in low temperature has important theroretical and practical significance to explore the mechanism of cold resistance.ADH genes play important roles in resistance to environmental stresses in plants including cold stress, it is important to isolate and identify of zoysia japonica ADH gene for a better understanding of zoysiagrass stress response.The stolon of bermudagrass and zoysiagrass were used, the dynamic changes of physiological and molecular indexes were measured under cold stress, and the objective of this study was to investigate the effect of physiological and molecular aspects under cold stress. Zoysiagrass were choosed because of its strong cold resistance, primers were designed to clone the full-length sequence of Zoysia japonica ADH gene, named as ZjADH, sequence analysis and functional confirmation of the gene were done. The main results are as follows:1. Turfgrass quality declined under cold stress, and the chlorophyll content, carotenoid content, photochemical efficiency decreased gradually. Proline, the soluble sugar, and the soluble protein accumulation gradually. Electrolyte leakage and malondialdehyde content increased gradually., The LT50 temperature of zoysiagrass and bermudagrass reduced under low temperature treatment, damage index evaluation results show that the cold resistance of the zoysiagrass is stronger than the bermudagrass.2. With the increase of cold stress time, the content of hydrogen peroxide and superoxide of zoysiagrass and bermudagrass increased gradually, the activity of super oxidase dismutase and peroxidase increased gradually too, and the activity of ascorbate peroxidase increased at first and then decreased. Cold stress promoted the expression of CytSOD, MnSOD, POD and APX, cold stress inhibited the expression of CAT and ChlSOD, the expression of FeSOD decreased at first and then increased.The study showed that, the plant started the antioxidant mechanism, scavenging active oxygen and reduce palant damage under cold stress.3. The content of abscisic acid in zoysiagrass and bermudagrass increased at first and then decreased, while the content of cytokinin decreased gradually under cold stress. With the increase of stress time, the expression of LEA increased gradually, and the expression of RAB increased at first and then decreased. The changes of endogenous hormones and the expression of low temperature induced proteins play an important role in activating the response mechanism and increasing the cold resistanceof plants.4. Two RACE reaction primers were designed for 3’RACE and 5’RACE to obtained 765bp and 816bp fragments. Full-length cDNA sequence of the gene is obtained by splicing sequence analysis, including a complete open reading frame 1140-bp in length, encoding a polupetide chains containing 379 amino acids. Blast comparison of amino acid sequence and phylogenetic tree analysis showed that ADH from Zoysia japonica is most closely related to the orthologue from Zoysia matrella. Those results displayed that ADH gene from Zoysia japonica was isolated successfully, named as ZjADH, and was submitted to GenBank (Access No.:KT596065).5. The relative expression of ZjADH in different tissues of zoysiagrass was analyzed by real-time quantitative RT-PCR method. The results shows ZjADH gene is expressed highest in roots, while the weakest in leaves. When induced by low temperatures, drought, and high salt stress, the ZjADH levels are increased, and those results displayed that ZjADH gene may be involved in response to ABA, MeJA and SA signal.6. The coding domain sequence of ZjADH gene was inserted into 3302-YFP vector, resulting fusion protein expression vector, ZjADH-YFP. Transformed tobacco mesophyll cells with a confocal microscopy revealed that the protein was localized in the cytoplasm.7. Construction of the plant expression vector 3302-ZjADH, transferred into Agrobacterium tumefaciens GV3101, then transformed wild-type Arabidopsis plants by floral dip method. Seedlings of glufosinate-tolerance were selected by sprayed with glufosinate. Genomic DNA was extracted from leaves of regenerated plants and PCR was performed. The results showed that transgenic plants were obtained. Total RNA of transgenic plants were isolated and analyzed by RT-PCR. The results showed that in transgenic Arabidopsis ZjADH gene, can be highly expressed.8. Tolerances to abiotic stresses analyses showed that the cold-tolerance of transgenic Arabidopsis plants was significantly higher than wild-type Arabidopsis thaliana. The expression levels of genes related to stresses in transgenic Arabidopsis were significantly higher than those in wild type Arabidopsis plants. The physiological and biochemical analyses of transgenic plants showed that the proline content of transgenic plants was higher than wild-type Arabidopsis.These results show that, zoysiagrass and bermudagrass increased freezing tolerance via the adjustment of osmotic,antioxidant defense,honnone and low temperature induced protein during cold accumulation, and the cold resistance of the zoysiagrass is stronger than the bermudagrass. Zoysiagrass were choosed because of its strong cold resistance, we cloned Zoysia japonica ADH gene of 1 MObp in length, localized in the cytoplasm, with highest expression in roots and induced expression levels under stresses. The transgenic Arabidopsis plants were successfully obtained and transgenic Arabidopsis showed enhanced tolerance to low temperature compared with the wild type, indicating Zoysia japonica ADH gene plays roles in plant stress responses and resistance to cold stress. |
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