Construction and analysis of Polygonum multiflorum cDNA library / 中草药

ABSTRACT

Objective To construct a cDNA library of three-year old Polygonum multiflorum leaf tissues so as to further research the gene regulation of secondary metabolite biosynthesis of medicinal plants. Methods Total RNA from leaf tissues of P.multiflorum was extracted and mRNA was purified,which were synthesized to double strand cDNA through reverse transcription.After the cDNA termini was blunted,the 5' end of EcoR Ⅰ adapters phosphorylated was conjoined,and then digested by Xho Ⅰ,cDNA fragments were fractionated by Sepharose CL-2B spin column.The fragments longer than 400 bp were linked to Uni-ZAP XR vector.The primary cDNA library was established after the recombinants had been packaged.Uni-ZAP XR Vector might fleetly release pBluescript SK-phasmids at the presence of ExAssist helper phage of coinfection and inverted E.coli SOLR.Finally,PCR and double enzymes digestion were used to analyze the range of inserts,respectively. Results The titer of cDNA primary library was 1.07?106pfu/mL and the length of exogenous insert was at about 0.5-2.0 kb with 5.4?105 recombinants,the recombinants of amplified library were 4.25?1011 and the rate of recombination was 98.5%. Conclusion The results indicate that the cDNA library of P.multiflorum leaf tissues has enough volume for screening the desired genes and sets up a basis for studying on gene regulation of secondary metabolite biosynthesis of medicinal plants besides.