| Potato(Solanum tuberosum L.)is an economically important food crop in the world and China. Potato tuber dormancy and sprouting have important influence on potato cultivation and process. The research about potato tuber dormancy and sprouting mostly concentrated on the storage physiology, genetics and cell biology. Low temperature or chemical inhibitors were mainly used to control the storage tubers sprouting in production practice. However, low temperature storage will cause breakdown of starch and accumulation of reducing sugars, using chemical inhibitors will increase people's concern about environmental and economic. Now, the mechanization of tuber dormancy and sprouting have not been understood absolutely, and the development of controlling technology was slow, which restricted the full use of potato seed and industrial raw materials of processing. The research about molecular mechanism and controlling measures of tuber dormancy and sprouting has important role on potato cultivation, fresh-keeping and industry development. The aim of the studies reported in this paper was to isolate and clone the potato tuber dormancy and sprouting related genes to look into molecular mechanism associated with dormancy and sprouting and establishing theoretical Basis and techniques for further study. The main results obtained are listed bellow:1. A subtractive library of potato tuber dormancy and sprouting related genes was constructed using suppression subtractive hybridization (SSH). Preliminary screening and Sequencing results showed that 310 valid ESTs were enriched. According to BLASTn and BLASTx analysis, 34 ESTs had never been reported function. the remaining had significant homology fragments or putative function with GenBank ESTs.2. The results of analyzing the tuber dormancy and sprouting related fagments revealed that it involve in a series of biological process including to the carbohydrate metabolism, protein metabolism, energy metabolism, nucleic acid transcription and translation, signal transduction, membrane transport as well as cell division and growth. By homology search and gene ontology analysis, the identified ESTs were classified in cell component, biological process and molecular function on level 2. Real time-PCR analysis of 13 transcripts identified as differentially expressed between tuber dormancy and sprouting revealed they were up-regulation or down-regulation genes at the process of potato tuber dormancy and sprouting.3. An ARF homology EST sequence was selected from the suppression substractive hybridization cDNA library between potato dormancy and sprouting library. The cDNA sequence of ARF gene which was obtained by silicon cloning belongs to potato ARF1. The tissue-specific expression of ARF gene was analyzed by real-time PCR and the highest expression level of ARF was detected in potato tuber.4. The relative expression of ARF gene in the potato tubers were analyzed by RT-PCR. These potato tubers were stored at 20℃and 4℃respectively, the gene expression of ARF were analyzed in different stages: for one week, four weeks, potato bud breaking, 2 mm, 5 mm and more than 10 mm long buds. The results showed that there was a increase-decrease-increase trend in the ARF gene expression of potato tubers during the process of removing from dormancy. Bud break and bud more than 10 mm long are two turning points. The decrease-increase trend in the expression of ARF gene was found in the potato microtubers which were harvested and kept in room temperature for 45 or 90 days when bud breaking or 2~3mm long. Bud breaking is a critical phase for removing from dormancy. The higher expression levels and the variation of expression of ARF gene in potato tuber demonstrated that the ARF probably participate in regulating the physiology of potato tuber dormancy and sprouting. |
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