山核桃脂肪代谢相关cDNA文库的构建

Construction of a fat metabolism-related cDNA library in Carya cathayensis

山核桃Carya cathayensis种仁含油量较高,油质多为不饱和脂肪酸。在差异显示反转录-聚合酶链式反应（DDRT-PCR）方法研究山核桃果实成熟时期基因差异表达的基础上,采用SMART（switching mechanism at 5’end of RNAtranscript）技术构建了山核桃脂肪代谢时期的互补脱氧核糖核酸（cDNA）文库。未扩增文库的滴度为5.0×105个噬菌斑形成单位（pfu）.mL-1,插入片段大小为0.7~2.5 kb,70.0%的片段超过1.0 kb;扩增文库的滴度为5.0×109pfu.mL-1,cDNA片段大小均在500 bp以上,重组率达94.5%,66.0%的片段超过1.0 kb。文库的构建为研究山核桃脂肪代谢机制、调控网络及相关基因的发现提供了条件。

Carya cathayensis is a species with a high oil content in its nuts.In addition,its oil is mostly unsaturated fatty acids.Based on differential display of genes during fruit maturity studied by DDRT-PCR,a fat metabolism-related cDNA library was constructed by SMART.Titering the unamplified library showed a titer of 5.0 × 105 pfu·mL-1,an insert size between 0.7 and 2.5 kb and 70.0% of inserts larger than 1.0 kb;and the amplified library had a titer of 5.0 × 109 pfu·mL-1,all of the inserts larger than 500 bp,and a recombination rate of 94.5% with 66.0% of the inserts longer than 1 000 bp.This work has laid a foundation for studies of fat-metabolizing mechanism and regulatory network as well as discovery of relevant genes.[Ch,7 fig.19 ref.]