Studies On Chilling Requirement And Dormancy Release Related Differentially Expressed Genes In Prunus Pseudocerasus Lindl.cv. Duanbing

Short-Petiole Cherry (Prunus. pseudocerasus Lindl.cv. Duanbing) is a promising variety of Chinese cherry (P. pseudocerasus) in Zhejiang aera. The control of bud break through a temperature-dependent mechanism is a key ecological factor in woody perennial plant survival. If bud germination can controlled artificially, it will bring economic benefit to fruit and horticultural industries. In order to extend cropping area, heated greenhouses were practically used in winter, and therefore, to predict dormancy release becomes a major factor in early fruit production. Furthermore, such concerns are likely to become increasingly important for field production in a period of rising global temperatures. Therefore, study the dormancy in flower bud of Short-Petiole Cherry is essential for standardization in field production and recognition the mechanisms of dormancy. In these studies, we took flower bud of Short-Petiole Cherry which is the most wide spread variety in Zhejiang area as material, studied the following questions related dormancy during dormancy period:1. The chilling requirement of Short-Petiole Cherry was calculated during two consecutive years of 2007 and 2008. The results showed that the dormancy of Short-Petiole cherry flower bud ends in late December. Based on the dormant progresses, using hydroponic culture method, 0～7.2℃,≤7.2℃and Utah model were used to calculate chilling requirement. The inter-annual comparison revealed Utah model results were relatively stable; the chilling requirement of Short-Petiole cherry flower bud was about 300 C.U. The data also indicated that in Jinhua area, the chilling accumulation in winter (November to January of the following year) was more than 800 C.U, which provided chilling reference for the introduction of other cherry cultivars to Jinhua aera in agricultural product.2. To unveil the physiolical process of dormancy, water contents, total phenol contents, soluble sugar contents, starch contents, soluble protein contents, free amino acid contents, ABA and GA₃ contents in the flower buds were studied in different phase of natural dormancy processs. The result showed that, the contents of water and soluble protein in dormancy release stage were increased in contrast to dormancy stage; while soluble sugars contents were decreased, indicated that the metabolism had been enhanced to establish the material base for germination. Changes of phenols and endogenous hormone contents were more relevant to the dormancy process. The contents of total phenols increased gradually in the primary stage of dormancy and decreased in the later stage, ABA content rose steadily in primary stage and reached its peak in early December, and then dropped. Contrary to ABA, GA₃ content was decreased in the primary stage of dormancy and increased in the period of dormancy release.3. mRNA differential display technique was used to analyse the differentially expressed cDNA fragments during the release of dormancy with the fulfilling of chilling requirement. In order to efficiently extract total RNA from flower bud of Short-Petiole Cherry, four differential methods were applied. By comparison, CTAB was confirmed to be the best method. Through differential analysis 79 differentially expressed fragments were cloned. Semi-quantitative RT-PCR was used to confirm results, and 18 positive cDNA fragments were identified as candidate genes related to dormancy release, which contained 11 up-regulated and 7 down-regulated gene fragments. Through sequencing and homology comparison, their putative function and roles in the process of dormancy release were analyzed and discussed. The up-regulated fragments showed high similarities at amino acid level to the following genes with known functions: ribosomal protein, leucine repeat protein kinase, ATP synthase subunit a, elongation factor la and starch metabolism-related enzymes et al; the down-regulated fragments included proline-rich protein, non-specific lipid transfer protein et al. Among these genes DNA-binding protein and antioxidative enzymes, the expressions of which were down-regulated, may play a key role in dormancy release.