Studies On The Differential Expression Genes Of Tea Plant (Camellia Sinensis) Seed During Cryopreservation

China is the place of origin of tea plant(Camellia sinensis(L.) O.Kuntze),and is abundant in tea plant germplasm resources.But due to natural and man-made factors,the loss of tea plant germplasm resources is of long standing.For long-term preservation of the genetic information of tea and the protection of tea plant germplasm resources,to find a proper way of long-term preservation of tea plant germplasm resources is of great importance.The seeds of tea plant,with its water contents varies from 35%to 40%,which usually be classified as recalcitrant type,are still of great difficulties in preservation.The traditional storage method is sand storage,that is stored in the environment of a certain humidity,temperature and ventilation conditions.But with the extension of storage time, the germination rate decreased rapidly,only about 20%after six months.At present,the main way of germplasm conservation is to preserve in low temperature germplasm gene banks.Among the existing problems,seed aging is the most important one,which present as the lower germination rate.The seeds of tea plants did not tolerate low temperature.When the seeds be stored in low temperature,the activity of the seeds can only remain a few months time.From the view of conservation of germplasm resources,it is far failed to meet the requirement.So far,the study of recalcitrant type seeds mainly focus on the relationships between storage conditions and seed germinability, and the study of physiological,biochemical and cellular changes during low temperature storage.But the study of the nature of recalcitrant type seeds' intolerance of low temperature is not enough.Using cDNA-AFLP technique,taking Longjing 43 clonal tea plant as materials,this experiment focusing on the researches as below:(1) The determination of critical moisture content of the seeds of tea plant(2) The study of differentially expressed genes of the seeds of tea plant during low temperature storageâ‘ Using cDNA-AFLP to study gene differential expression of the same cultivar after low temperature inducedâ‘¡Using RT-PCR to identify differentially expressed genes after sequenced of differential bandsâ‘¢Using RACE to clone full-length cDNA sequences and analyse sequence.Results show 8 differential fragments were gained;through NCBI BLAST,6 genes showed significant homolog to known genes according to the sequencing of 15 genes, including transport facilitation,transcription and energy-related;These differences in the expression of sequence fragments can be divided into six categories are:metallothionein FtsH(TDF1),the aging protein of seeds(TDF2),protein kinase(TDF3),human epidermal growth factor(TDF4),nucleic acid and protein transcription regulatory factor(TDF4),rice protein coding assumptions(TDF6).Differences in the expression of these fragments TDF1, TDF2 of tea seeds in cold storage in the course of the first was found.RACE use of technology has been TDF1 and TDF2cDNA full-length,respectively, for a total length of 709 bp and 917 bp.One TDF1 is metallothionein FtsH homology of high protein,TDF2 with the aging of Arabidopsis has a high homology.