目的 为进行药用植物次生代谢产物的生物合成基因调控研究,构建了三年生何首乌叶cDNA文库。方法 以何首乌成熟叶片为材料提取其总RNA;纯化出mRNA;反转录合成双链cDNA;钝化cDNA末端;连接上EcoR接头;经Xho酶切后,使用Sepharose CL-2B凝胶介质滤过,收集400bp以上的片段与Uni-ZAP XR载体连接,经包装蛋白包装成cDNA文库。Uni-ZAPXR载体可以在辅助噬菌体ExAssist helper phage的共感染下,快速释放出pBluescript SK-噬菌粒,经转染E.coliSOLR后,铺于氨苄青霉素抗性平板,分别利用PCR和双酶切的方法鉴定插入片段的大小。结果 测定该初始文库的滴度为1.07×10⁶pfu/mL,含有5.4×10⁵个重组子,插入片断长度为0.5～2.0kb;扩增文库的重组子为4.25×10¹¹个,重组率为98.5%。结论 经检测所构建的文库库容量满足基因筛选要求,为进行中药材相关功能基因调控研究奠定基础。

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.

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