Construction Of The Root Full-length CDNA Library For Trifoliate Orange And Bioinfomatic Research Of EST

Trifoliate orange (Poncirus trifoliate) is an important rootstock for citrus in our country, with cold tolerance, being resistant to or tolerant of excellent features a variety of disease. In order to understand the root gene expression of trifoliate orange and select genes against biotic or abiotic stress, we constructed a root full-length cDNA library, chose clones to sequence and analysed the ESTs trough bioinformatics methods. The main results were as follow:1. Total RNA was extracted from the trifoliate orange root with TRIZOL. The double-stranded cDNA was obtained through reverse transcription and by LD-PCR, then was digested with restriction endonuclease Sfi I. Bing removed short fragments less than 500bp through CHROMASPIN-400 column, the mixture of long fragments were connected with the vector pDNR-LIB, and transformed into Escherichia coli JM109 through heat shock. The original full-length cDNA library was acquired with the reorganization frequency of 95% and storage capacity 1.08×106pfu.mL-1.2. The 400 clones were selected randomly from the trifoliate orange root full-length cDNA library and sequenced. Removed the vector and adaptor fragments,386 ESTs with the effective length more than 150 bp were got. The lenghth of these ESTs was from 150 bp to 1571 bp and the average was 560 bp. These 386 ESTs were clustered through BioEdit software and total of 178 Unigenes were got including 43 Contigs and 135 Singlets.3. After homology search, we found 125 Unigenes had homologous ones in the GeneBank database and 53 did not, with percentage 70.3% and 29.7% respectively. The gene functional annotation showed the biotic or abiotic stress-related genes were the most, amount to 15%. such as MLP43. dehydrin, dormancy-associated protein, DYL1, PIP2.1, PRXR1, Aspartic proteinase. Lea5. aldo/keto reductase. dnaJ, metallothionein. Calmodulin 1 and etc.4.36 Unigenes had a complete open reading frame,5'and 3'terminal untranslated region analysed with ORF Finder software. Among them,24 full-length genes had homologous ones in the GeneBank database through tBlastX analysis and the other 12 did not have. On this basis, for six full-length genes of protein function prediction.