Cloning And Prokaryotic Express Of Fls Gene From Eucommia Ulmoides Oliv.

As an exclusive species of China, Ecommia ulmoides of Eucommiaceae has been listedin the species of national second-class protective plants. It is a multipurpose tree species,which was used in the medicine as the main function, so it obtains high value for developmentand utilization. Flavonoids extracted from Ecommia ulmoides are important activecomponents.FLS is one of2-oxoglutarate-dependent dioxygenase family, and the fuction is to catalyzedihvdroflavonols into flavonol by forming the double bond. Pharmacological effects offlavonol are fall blood sugar, blood fat, anti-aging, removal of oxygen free radicals and so on.The conserved cDNA sequence of Fls was amplified from total RNA of leaf tissue byreverse transcription-polymerase chain reaction (RT-PCR), and rapid amplification of cDNAend (RACE) was used to obtain the full-length cDNA of Fls. The Fls of open reading frame（ORF）was digested with restriction endonucleases, recombined into pET-28a（+）．Thevector of the pET-28a-Fls was constructed and expressed in E.coli BL21(DE3). The mainresults are as follow:1. A pair of degenerate primers was designed based on the genes of Fls and conservedregions of reported FLS amino acid sequences, and it was used to amplify conservedsequence of Fls by reverse transcription-polymerase chain reaction (RT-PCR) from Ecommiaulmoides, and RACE was used to obtain the full-length cDNA of Fls. The full-length cDNAof Fls is1200bp(GenBank accession No. KC447296), which includes1011bp of ORF, and3’ the end of the full sequence has the the obvious poly(A) tail signal.2. Using the DNAMAN to analyse the amino acid sequences of FLS about13species,the results show that identify of the C terminal region is higher, while the C terminal is lower;Phylogenetic trees and evolutionary distance of the deduced amino acid sequences of plantFLS were established by Clustalx2and MEGA4, results show that eucommia ulmoides isclose to Nicotiana tabacum, P. hybrida，Solanum tuberosum, Lonicera japonica, and distantrelative with ginkgo.3. The analysis of the amino acid sequence reveals that Fls of Eucommia ulmoidesencoded336amino acids with molecular weight38.03kD and theoretical PI5.59. The proteinanalysed by Protparam is41.95of the instability index and10h of the estimated half-life in Escherichia coli.; The potential transmembrane of FLS is5times and FLS consists by randomcoil (34.52%), apha helix (32.74%), extrend strand (25.30%) and β-turn (7.44%); Last, thedomains of FLS are two which were DIOX_N and2OG-FeII_OXY.4. Fls was constructed into expression vector pET-28a(+) for over expression inEscherichia coli BL21(DE3).The SDS-PAGE showed that the flavonol synthase proteins wereexpressed in Escherichia coli BL21(DE3), and its molecular weigh was found to be about44kD, nearly equal to the predicted.