Construction Of Forward And Reverse Subtracted CDNA Libraries From The Leaves Of Poncirus Trifoliata (L.) Raf. Under Low-temperature Stress And Bioinformatics Analysis

Citrus is a subtropical fruit tree which has been widely cultivated around the world. However, the plantation of citrus frequently suffers from low-temperature stress, and so the economic loss was huge and will continue at an ever increasing pace with the climate change. Yet Poncirus trifoliate (L.) Raf., an interfertile citrus relative, is extremely cold tolerant and can survive freezes at-20℃if fully cold acclimated. To investigate the differences and molecular mechanism of cold acclimation has been a major focus of research, especially after classical breeding techniques were found to be incapable of improving the cold tolerance of plants significantly. In addition, the recent accomplishment and release of citrus genome will facilitate the further genomic rearch of the underling stress tolerance mechanisms in citrus. Therefore, to acquire more genetic and biological information has far-reaching and significant impact on the functional research and further production of citrus.Based on the laboratory's former research experience in citrus, P. trifoliate was chosen as experimental material and treated in the environment which mimiced the temperature change during the period of ealier winter. Then suppressive subtractive hybridization technique was applied for the construction of forward and reverse cDNA subtracted libraries. Moreover, these expressed sequence tags (ESTs) obtained from constructed SSH cDNA libraries were analyzed by bioinformatics tools. The results indicate that ESTs are mainly related to genes involved in metabolism, cell structure, protein synthesis, signal transduction and other uncertain functions. In general, the results show that cold tolerance is a complex trait in citrus, which involves the coordination of many genes and multiple metabolism pathways. This research, on the one hand, proved and further optimized the application of SSH technique on citrus research; on the other hand, the construction of two subtractive libraries and identification of candidate EST sequences would facilitate further functional studies of cold-related genes and provide important insights into the molecular mechanisms of cold-stress tolerance and genetic breeding in citrus.