The Expression And Analysis Of Heat Shock Protein(HSP) In Tea Plant (Camellia Sinensis) Seed During The Cold Storage

Tea plant(Camellia sinensis) is a kind of important economic crops in China and plays an important role in the national economy.China is the origin of tea plant, where germplasm resources is extremely rich.In tea plant classification system of 37 kinds and three varieties,apart from C.pubicosta Merr,all the others are generated in china.Now we have used the method of planting saved for the preservation of tea plant germplasm resources,because the seed of tea is in the recalcitrant seeds storage behavior,which have cold intolerance and intolerance both dry.This means not only use up a lot of preservation of land and human resources,but also vulnerable to external environmental impact.In vitro culture technology,due to the need for long-term subculture which is not only expensive but also will cause genetic instability,and the high content of polyphenols,is far from achieved the long-term preservation of germplasm resources requirements in the current level of technology.The seed of tea is the natural reproduction of future generations,which stored the integrity of genetic information and is convenient to carrying,transporting and storage as the materials of preservation,playing an important role in multiplication and spread. It is very important to studying the significance of the storage of tea seed,because long-term preservation of seeds is the key to resolving the degradation.The traditional way for storage was by sand,that is stored in the environment of a certain humidity, temperature and ventilation conditions,but stored in this way the germination rate decreased rapidly with the extension of storage time.Six months later,the germination rate will be only 20 percent.For a long time,there are less study in this area.There are a few number of studies conducted a preliminary useful attempts on the preservation.But so far there are little study for seeds in the process of physical, biological and cytology areas,much less related to mechanism of molecular biology.cDNA-AFLP technique is a important tool in the research of transcription genomics which is developing in recent years.Because of its high reproducibility, accuracy,reliability and dispensation with sequence information,this technique is widely applied to analyse comprehensively and systematically the difference gene expression.This research faces the reality that the tea seed has intolerance of cold storage and take Longjing 43 which has high setting rate as the object,researching the gene expression spectrum difference performance characteristic which caused by cold stress through the cDNA-AFLP,and use the biological information method to forecast the structure and the function of the difference gene expression.With the resistant related unknown function sequence,RACE technology is used to clone its span DNA sequence.We hope this research could reveal the relevant molecular mechanisms of tea seed induced by the cold stress,and the results are following:(1) There were 10 fragments selected from the amplification with 64 primer-pairs that showed differential gene expression between the seeds stored in the low temperature and control the newly plucked ones.After sequence analysis,7 fragments were found to be highly homologous to GenBank sequences,including low molecular weight heat shock protein,myb family transcription factor,putative senescence-associated protein,F-box family protein,phosphodiesterase,protein kinase and 1,4-alpha-glucan-maltohydrolase,the other 3 fragments were unknown or no significant similarity found.(2) Through using the RACE technology,we has obtained TDF3 cDNA span,and the span is 819bp.The protein encoded by TDF3 has the very high homology to the low molecular weight heat shock protein and the ORF of it is 453bp which codes 150 amino acids.