Construction Of Ripening Fruit Epicarp And Pulp Subtractive CDNA Library And Preliminary Analyses Of EST Sequences Of Ponkan

Ponkan(Citrus reticulata)is a late-maturing variation from ordinary ponkan in China.Its maturity is 40～60 days later than ordinary ponkan under the same conditions.In addition,it has relatively higher cold-tolerance and high quality fruit.It is of great importance in breeding and production for high quality fruit to study the mechanisms of hihg fruit quality and late maturation and the discovery of the corresponding genes.This study used the materials of fruit peel and pulps of Yanxiwanlu and the technique of suppression subtractive hybridization(SSH)to enrich the diffeeentially expressed transcripts to construct the corresponding subtractive cDNA libraries.The peel-and pulp-specific ESTs were obtained through sequencing and classified based on analysis of their anotations,which provides the basis for further studies such as the discovery and utilization of the elite genes.Peel and pulps were collected from mature fruit of Yanxiwanlu.Conventinal total RNA extraction method using Pehnol/SDS/LiCI was modified to extract RNA.High quality mRNA was isolated and reverse-transcrpted into cDNA.Clontech PCR-SelectTM cDNA Subtractive Kit was used to construct the cDNA library by following the instructions of manufacturer.Briefly, two rounds of subtractive hybridization were first performed.The differentially expressed cDNAs were then exponentially amplified by suppressive PCR.PCR products were further purified and ligated into vector pTZ57R/T.The plasmids were then transformed into competent cells of E.coli strain DH5α.A total of 9784 clones were obtained,of which 87 were blue clones. Therefore the recombination rate of the clones was 99.1%.A total of 1019 white clones were randomly picked and cultured to constitute a subtractive cDNA library.The sizes of the inserts were between 100 and 500 bp as estimated by PCR bands of 100 randomly picked clones,which was in agreement with the expected values.So far 770 clones were sequenced and 411 ESTs were obtained.The search against GenBank revealed that 377 ESTs had significant similarities with known genes.One highly expressed EST was found to have thirty one occurances in the library.Another highly expressed EST had twenty-eight occurnces.One EST appeared twenty-five times.Five ESTS appeared between ten and twenty times.Fifty five occurred between two and ten times.Annotation of the gene function showed that these ESTs were mainly involved in pigment synthesis,energy metabolism,primary metabolism,secondary metabolism,defense against strsseses,signal transduction,transcription, flavor formation,oil metabolism,peel softening,antioxidant metabolism et al.There were 34 ESTs without known functions.