CDNA Library Construction Using Floral Orgrans, Fruitlets Of Ponkan Mandarin And Expressed Sequence Tags Analysis

The generation of expressed sequence tags(ESTs) has been proven to be a rapid and approach by which to identify the novel genes.In order to identify and isolate of Ponkan, a cDNA library using floral organs and fruitlets of Ponkan was constructed,and it was subjected to 5' EST sequencing and bioinformatical analysis.The main results were as follow:1.Total RNA was isolated from floral organs and fruitlets of Ponkan using improved method of guanidine isothiocyanate,total RNA was separated and further purified to obtain mRNA(messenger RNA),the latter was synthesized to the first strand of cDNA with RT(Reverse Transcriptase).Further changed to double-cDNA strands was realized by using"nick-translate"method.The cDNA was then fractionated CHROMAS PIN-400 and the fragments longer than 500 bp ligated to the vector pDNR-LIB to transform the host by electroporator.Finally the cDNA library using floral organs and fruitlets of Ponkan was constructed.The sink size of the cDNA is 1.13×10~8,in which 97%phages were recombinant,insert sizes ranging from 0.5-2.5 kb were obtained.The results indicated that the library was integrated and efficacious,which is suited for ESTs sequencing.2.Totally 503 clones were randomly selected from cDNA library of flower and fruitlets of Ponkan and partially sequenced,487 readable sequences that are more than 100 bp were produced by analysis with CAP3 software.And the average length of the 487 ESTs was 522 bp,the average quality is 37.06,the GC content is 40.49%.Using the CAP3 software,the 487 effective ESTs has been cluster analyzed and assembleed into 298 UniGene including 27 contigs and 271 contigs were composed of only one EST which was called singlet.3.Unigenes has been functionally classified based on Gene Ontology.The genes have been annotated functions according to GO three classification levels(the molecular function,biological process and cell component).Genes with function annotation or putative function annotation were assigned to 11 categories according to the classification of BlastX.They were genes related to metabolism(corresponding 21.02%of total genes), proteins ynthesis(19.89%),energy(8.52%),Transporters(9.09%),Transcription (14.20%),cellular signal transduction(4.55%),Cell growth and division(3.98%),Disease and defence(8.52%),Cell structure and Cellcycle(3.41%),secondary metabolism (3.41%),protein destination and storage(3.41%).