| Lily is one of the world famous bulb flowers, Natural dormancy is a characteristic of lily,Bulb dormancy can be completely broken after a period of low temperature treatment,and it will Normally flower and grow next year. Bulb dormancy-breaking is a complex biological process,Hormones,carbohydrates and the internal and external environmental factors and genetic constitute a complex network system to control the whole process of the dormancy.the research on dormancy mainly concentrated in morphology and physiological and biochemical changes during breaking dormancy under low temperature. Less in molecular level. We studied cytological and physiological changes of bulbs in Lilium pumilum during breaking dormancy under 5℃. On the basis of these research results, We constructed 2 SSH-cDNA libraries using dormant and dormancy breaking bulbs. a lot of dormancy regulation related genes were isolated, and we isolated differentially expressed proteins and analyzed their functions Using two-dimensional electrophoresis technology in the process of breaking dormancy under low temperature. In order to lay the foundation for further study bulb dormancy mechanism. The main results were as follows:1.Using paraffin section and stereoscopic microscopic observation, during cold storage, the lily flower bud differentiation is mainly divided into four periods:floret primordium differentiation stage, outer perianth primordium differentiation stage, inner perianth primordium differentiation stage, stamen and pistil primordium differentiation stage. the number of starch grains gradually reduced in Scales and apical bud cells. the number of fission cells increased gradually. after 36 d,Transmission Electron Microscope observation showed that the material and energy metabolism of bulbs were weak between 0 to 12 days and bulbs couldn’t sprout; the germination rate of bulbs was lower and mitochondria and golgi vesicles increased,Cells exchanged materials through the plasma membrane endocytosis and plasmodesmata between 12 to 48 days. Nucleoli were incompact and endoplasmic reticulums scattered orderly between 48 to 72 days, and endoplasm bridges formed at the edges of vacuole membrane and plasma membrane, with some of them extending to the nuclear envelope,Intracellular and intercellular connection had been established in this period. Bulbs appear new root;the apical bud has been extended to the top of the bulb after 84 d,Endoplasm bridges and the plasmodesmata connected in budding period, and the internal structures and quantity of organelles increased obviously,Dormancy is completely broken in this period.the apical bud elongated gradually during refrigerated condition. The respiratory rate showed a trend of decline after rising first. Cold storage time longer, shorter bulb germination time.2.The obvious changes of nucleic acid content in apical buds and basal plates of Lilium pumilum bulbs.The change of DNA, RNA content and RNA/DNA ratio in apical buds were most obvious when bulb refrigerated 36 d,36 d is a turning point of the total amount of nucleic acid, DNA in inner and outer scales.the content of soluble protein in apical buds and basal plates increased within 0～36 d during refrigerated condition. the content of soluble protein in different parts of the bulbs reduced rapidly after 48 d, which was the important symbol of bulb for breaking dormancy. the sugar metabolic activity was strong in the process of dormancy breaking, the starch content of bulbs decreased, the activity of amylase activity remains high.At the beginning of the storage, the sucrose content, sucrose metabolism key enzyme SS and SPS were on the rise, there was a synergy between SPS and SS in controling of sucrose metabolism and transport. During refrigerated conditions the metabolism direction of starch to soluble sugar promoted the increase of AGPase activity. AGPase is likely to be a important physiological deciding factor for dormancy breaking.SOD, POD, CAT activity in the early stage of the cold storage has a downward trend, during middle and later periods, SOD, CAT and ASP are elevated, POD activity of scales continued to decline in the process of cold storage, POD and PPO activity of apical buds and basal plates is also on the rise in the middle of the cold storage. The role of various metabolism related enzymes in different organs is not the same.,36 d is the starting point of bulb breaking dormancy, at 60 d bulb dormancy basicly broke.3.We constructed 2 SSH-cDNA libraries using dormant and dormancy breaking bulbs of Lilium pumilum. The forward library contained genes preferentially expressed in dormancy breaking bulbs and the reverse library contained genes preferentially expressed in dormant bulbs. 100 positive clones were selected respectively for sequencing from the two libraries. We analyzed ESTs in BLASTx of NCBI and unigenes positioning in the pathways using KOBAS system. Results indicated that in dormant bulb library, these functionally known genes were detected in stress response. Phenylalanine metabolism and MAPK signaling pathway were associated with dormancy. In bulb dormancy breaking library, GO function of different genes mainly enriched in protein synthesis, cell resistance and defense, signal transduction, binding activity, transcriptional activity and metabolic process, etc.the function of ESTs involved in sugar metabolism, hormone response and signal transduction,Linoleic acid metabolism and Starch and sucrose metabolic pathway was involved in dormancy breaking process. the establishment of both libraries laid a foundation for further understanding the molecular mechanism of lily during bulb dormancy.4. A total of 31 differentially expressed proteins were separated by using fluorescent difference gel electrophoresis, Among these proteins, there are 14 proteins up-regulated and 17 proteins down-regulated in the dormancy breaking bulbs. up-regulated proteins accounts for 45.16% of the total,and down-regulated proteins accounts for 54.84% of the total, These Proteins were classified by function into 6 categories. Comprehensive analysis shows that these proteins are mainly stress protein, may involve metabolic process of the bulbs, and regulating the bulb dormancy breaking. |
PDF Full Text Request