Construction Of CDNA Library Of Bark In Ramie(Boehmeria Nivea(L.)Gaud) And EST Analysis

Ramie(Boehmeria nivea(L.)Gaud),a perennial herbage crop is an important source of cellulose-rich naural fibres(bast fibres).It's primary purpose is making clothing.Around Changjiang river,Ramie has 3 crown of the year,the frist-crop,the second-crop and the third-crop.We have been interested in the diversity of genes expressed during ramie fibre cell wall formation, with the aim to understand fibre development,The constructed 2 cDNA libraries derived from the bark of the second-crop and the third-crop.The second-crop cDNA library was used for EST sequencing and bioinformation analysis.The main results .were as follow:1.Total RNA was extracted from the ramie(Boehmeria nivea(L.)Gaud)) barks harvested at the mid-growing stage, and mRNA was purified.After double-stranded cDNAs synthesis,EcoR I-Sma I adaptor were added to blunted cDNA and digested with Not I ,and fractionated by spin column.The fragments longer than 400bp were ligated into the pAP3neo predigested Vector.The cDNA libraries consisted of 2.2×10~5cfu/ml and 1.7×10~5cfu/ml, a majority of inserts size ranged between 500bp and 1500bp.2.384 ESTs were produced form randomly selected clones of the second-crop cDNA library,275 useful ESTs that are more than 150bp were identified by analysis with Cross-match software,the ratio is 71.61%.3.The 275 ESTs were identified by BlastN searches against the NCBI non-redundant nucleotide databases and BlastX searches against NCBI non-redundant protein databases,36% of the total 275 ESTs showed significant homology with the nucleotide databases and 72.73% with the protein databases.4.These ESTs were classified into 12 different categories,the hightest proportion of which was related to protein synthesis,17.5%,and the others were Energy(13.5%),Cell division(0.5%),Signal transduction (10.5%),Metabolism(8%),Secondary metabolism(l%),Protein destination and division(4%),Transporter (10%),Disease/defence(7.5%),Transcription(13%),Cell structure (6.5%),Unclear classification(8%). 5.In 12 categories,there is 15.3% of the total sequences were high expressed abundance genes(espression frequency≥4),7.3% were moderate expressed abundance genes(2≤espression frequency<4),5.1% were fibre development-related genes.