Construction Of Normalized CDNA Library And Analysis On Expressed Sequence Tags Of The Flower Development Of CCRI 36

The generation of expressed sequence tags(ESTs) has been proved to be a rapid andefficient approach to identify the novel genes and analyze the function of genes.In order to identify and isolate those genes related to the flower development,a normalized cDNA library of flower development of zhong36 was constructed,and it was subjected to high-throughout 3'EST sequencing and bioinformatical analyzing.The main results were as follows:1.Total RNA was isolated from different time of flower development using improved method of CTAB,and total RNA was separated and further purified to obtain mRNA(messenger RNA),then the latter was synthesized to the first strand of cDNA by RT(Reverse Transcriptase) and further changed to double-cDNA strands by using "SMART" method.The cDNA was then normalized by DSN and the fragments being longer than 500bp were renovated by spin-400 and transformed to the host by ligating them to the vectors with electroporator.Finally,the primary normalized cDNA library of tissue of growth of flower was constructed.The sink size of the primary cDNA is 1.7×10~6,in which 100% phages were recombinant,and the insert sizes ranging from 0.5～3.0kb were obtained.The results indicated that the library was integrated and efficacious,which is suited for ESTs sequencing.2.Totally 3421 clones from the normalized cDNA library were randomly selected,and 3175 readable sequences that are more than 100bp were produced by analysis with Phrad software.the average length of the 3175 ESTs was 486bp.3.Using the Phrap software,the 3175 effective ESTs has been analyzed by cluster and assemblied into 2906 UniGene including 233 contigs and 2673 Singlets explained with composing of only one EST.4.In 3175 sequences,84.19%of the sequences appear only once,15.65%of the sequences appear for 2-5 times,and only one sequence appears for 5 times.BLAST analysis of this high-copy sequences shows that it has high homology to the EST nucleotide database,but the product of this sequence is still unknown.At the same time,the level of continued expressed gene for investigating the balance effect(like actin and ribosomal protein gene) drop to less than 3 copies,which indicate the proportion of high abundant genes have decreased,and the proportion of other gene has increased after normalization.The effect of normalization of the library is significant.5.The 3175 ESTs were blasted against the non-redundant nucleotide and protein database of NCBI.On the basis of NCBI database searches,31.73%of the total 913 ESTs showed significant homology with the nucleotide database and 26.52%with the protein database.There are nine ESTs that have high homology with MDS-BOX genes.6.According to the accession in Swissprot database,the unigene has been functionally classified based on Gene Ontology.The 763 genes with homology alignment have been annotated into different 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 16 categories.They were genes related to Metabolism(23.92%),Translation(20.18%),Posttranslational modifcation,protein turnover,chaperones(15.73%),General function prediction only(11.93%),Energy(6.82%),Transcription(4.06%),Signal transduction(3.67%),Function unknown(2.75%),Secondary metabolism(2.62%),Chromatin structure(2.36%),Cell structure(1.57%),Transporters(1.44%),Replication,recombination and repair(1.31%),Cytoskeleton(0.92%),RNA processing and modification(0.39%),Disease and defence(0.26%).