Construction And Preliminarily Analysis Of Suppression Subtracted CDNA Library From Drought Stressed Citrus Leaves

Drought is one of the most important environmental stresses that impact plant growth and crop productivity.Citrus is one of the most important fruit crops in the world,the yiled of which is dramatically limited by drought stress.It is impotant to study the drought-resposive mechanism at the molecular level in rder to improve drought resistance in Citrus using biotechnology.In this research,a suppression subtractive hybridization(SSH)cDNA librany was constructed using leaves of drought stressed Citrus.reticulatacv ponkan plants with the purpose of enriching drought-induced genes.Drought stress treatment was carried out by placing plants under shelded greenhouse without watering for 20 d.Control plants were watered normally.Total RNA was isolated from leaves using Phenol/SDS method with some modification,mRNA was isolated by using Oligotex mRNA Mini Kit.The drought-induced cDNAs were obtained by using commercial SSH cDNA library construction kit and were ligated into pTZ57R/TVector.Ligated products were transformed into E.coli cell line DH5α.A cDNA library containing approximately 438 individual recombinant clones was thus obtained.The quality of the library was satisfactory as revealed by PCR amplification of the inserts.Eighty five clones were randomly selected and sequenced.A total of 78 EST sequences were obtained.Comparasion of the EST sequences showed that 62 of them were different.Blast search of the ESTs against those in Genbank showed that around 90%of the ESTs shared high homology with known ESTs,of which more than 80%were homologous to those originated from the cDNAs induced by abiotic and biologic stresses such as pathogen infection and drought,heat,salt,cold stresses.Some ESTs were highly homologous with those induced by more than one stresses, indicating that different stress responsive pathways share some common mechanism in plants.The deduced products of the 51 ESTs with known functions could be claassified into eight categories: metabolism,trascription,defense,photosynthesis,protein synthesis,signal transduction,unknown function.Among them,28 genes were drought stress-related genes,and their function including signal transduction and gene expression regulation,substance synthesis,defense,active oxygen elimination,cell defense,protein synthesis,and other physiological and biochemical pathways. Many drought resistant related ESTs were obtained putative calcium-dependent protein kinase,translation initiation factor,dehydrin DHN6,superoxide dismutase,NADP-dependent malic enzyme,heat shock protein and Myo-inositol-1-phosphate synthase and many other crucial genetic information.