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Construction and ESTs Analysis of the Normalized cDNA Library from Chrysanthemum Cinerariifolium Flowers

Pyrethrum(Chrysanthemum Cinerariifolium) is a species of perennial herbage plants, and a sole industrial insecticidal plant all over the world. Because of excellent insecticidal characteristics of pyrethrins, including high efficiency, low toxin, broad-spectrum, low residues, and a low impact on the human being and environment, pyrethrins have become one of the most important biologic insecticides.The pyrethrins are mainly synthesized in pyrethrum flowers and the content is more than 95%. The content in the dried flowers is about 1.2-2.0%. At present, natural pyrethrins have been widely used in agriculture, forestry, biological pesticides, food hygiene and safety and other fields. Improving the content of pyrethrins in pyrethrum through genetic Engineering technology is restricted, for little is known about pyrethrins-related genes. The cDNA library from pyrethrum will provide a basis for cloning and characterization of genes related to the synthesis and metabolism of pyrethrin.Using pyrethrum flowers as material, the library has been constructed in this study. We selected and sequenced partial colones, compared the homology and analyzed the gene function by bioinformation method. The main results were as follows:1 . The total RNA of pyrethrum was extracted with RNA extract kit. The agar electrophoresis show that the total RNA has two bright bands and the ratio between the two bands is 2:1. The purity fit for following experiments.Using SMARTⅣOligonuleotide and CDS-3M adapter as primer, PowerScript Reverse Transcriptase as polymerase, first-strand cDNA is synthesized; and following ds-cDNA is synthesized by LD-PCR. After PCR purification, DSN and sfi I process, small cDNA fragment and some rRNA are removed. Then cDNA fraction is ligated into pDNR-LIB vector and transformed into E.coli JM109. The cDNA library obtained is stored at -80℃after amplification.2 . Dilute the cDNA library with LB/Cm medium. The mixture is poured onto 90 mm LB/Cm plates which are prewarmed at 37℃, and then the plates are inverted and incubated at 37℃overnight. The titer of the library is 2.8×10~5 pfu/mL, and recomninant percentage is 96%. The result of PCR with M13 primers show the length of insert fragments are between 400 bp and 1 300 bp. The titer of amplified llibrary is about 2.0×10~9 pfu/mL.3 . Partial clones are selected from the library. The result of PCR and 1.2% agar gel electrophoresis show that most of the colones have inserting fragments. 65 positive colones are sequenced with T7 and 58 ESTs are obtained finally.4 . The 58 ESTs are blasted against the non-redundant nucleotide and protein database of NCBI, and 48 of the total 58 ESTs show homology with sequences registered in these databases. Out of these, 40 ESTs have a definite annotation, 8 ESTs are unnamed and hypothetical protein, the other 10 ESTs have no significant similarity.