| Zoysiagrass (Zoysia japonica Steud.) is a kind of turfgrass with great potential applications. Especially in China, the resource of zoysiagrass lines for the first in the total of the world, and also it is suitable for the regional wide with strong resistance, low cost of maintenance and developed the underground root system. Therefore, it plays important role in the modern construction of urban greening and playgrounds, and even in water and soil conservation. However, the disadvantages of zoysiagrass as short green period has become the bottleneck of its application. In this study, aim for the extension of green stage in zoysiagrass, we found GA3 could alleviate leaf senescence induced by darkness, and separated GA3 regulation related gene, which gives more understanding on antiaging mechanism in GA3 pathway, and also is important theoretical and practical value.Here, we first show that zoysiagrass seedlings treated with GA3 could delay the leaf senescence induced by darkness. The zoysiagrass seedlings subjected to darkness manifested in sharp chlorophyll and protein loss, improved contents of MDA and H2O2, also exhibited higher cell death folds. However, the seedlings treated by GA3 exogenous before darkness stress displayed better phenotype than control. Although mechanism of GA3 on leaf senescence is unknown, it is first time reported that the GA3 plays negative role in the leaf senescence of zoysiagrass.To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-GA3-treated and GA3-treated seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Total 22 differentially expressed genes selected by subtractions were classified into five categories according to their putative functions generated by BLAST analysis, they were "General metabolism", "Signal transduction", "Cytoskeleton"; "Sequences without identity" and "Unknown function".Expression of five selected genes, OMT, SAMs, V-ATPase, CRY2 (gene of cryptochrome 2) and An were examined by RT-PCR, when the zoysiagrass were under normal senescence and senescence induced by environment. RT-PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence. Especially, the CRY2 exhibited higher expression in the young leaves, and up-regulated on the later stage of environment stress, and also display interested expression pattern under GA3 exogenous and darkness stress, so the CRY2 is selected for further research.The full length cDNA of CRY2 was obtained by RACE-PCR, and the protein encoded by CRY2 was analyzed by bioinformatics. The results showed that CRY2 belongs to the family of cryptochrome with three conserved functional domain such as DNA photolyse, FAD-binding superfamily and crypochrome C terminal superfamily. In addition, CRY2 showed high homology with OsCRY2 in rice and the phylogenetic analysis demonstrated similar pattern. Therefore, CRY2 was regarded as the ZjCRY2, a gene of cryptochrome 2 in zoysiagrass. Besides the bioinformatics analysis, the investigation on the senescence of Arabidopsis cry2 mutant was carried out, and the results also proved the CRY2 (AtCRY2) could participate in the regulation of developmental leaf senescence.Further, the over expression and RNAi expression vectors of ZjCRY2 were constructed for transformation. After embryogenic calli were pre-cultured, they were genetic transformated by biolistic bombardment. For about 4 months of subculture, the transgenic calluses with hygromycin resistance were obtained, and the transgenic seedlings would be obtained after another two months.In this study, five genes associated with leaf senescence in zoysiagrass were found through the SSH. The expression patterns of these genes were analyzed, and the CRY2 was selected finally and regarded as the functional gene related to GA3 and darkness crosstalk on the leaf senescence. Also, we obtained the full length cDNA of CRY2, and confirmed the CRY2 is the gene of cryptochrome 2 in zoysiagrass as the ZjCRY2 by bioinformatics analysis. The function of ZjCRY2 associated with leaf senescence was further predicted by analysis of Arabidopsis cry2 mutant. Finally, the vectors of over expression and RNAi expression of ZjCRY2 were constructed, and the transgenic calluses were obtained. All the results in this study, could contribute to the further research on the GA3 signal pathway related to leaf senescence and the application of transgenic turfgrass and ornamental plant with enhanced resistance to leaf senescence. |
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