红花种子cDNA文库构建及脂肪酸代谢相关基因的克隆

Construction of safflower cDNA library and clone of fatty acid metabolism genes

【目的】构建红花种子cDNA文库,挖掘红花脂肪酸代谢途径相关基因,为红花品质改良的基因工程研究奠定基础。【方法】以红花成熟种子为材料,通过Trizol法,提取红花种子总RNA,使用GeneRacer?Kit和CloneMinerⅡcDNA Library Construction Kit试剂盒构建cDNA文库。并对cDNA文库的重组率和插入片段进行鉴定,从中获得contig和singlet片段及脂肪酸代谢相关基因,通过荧光定量PCR,测定delt-12脂肪酸脱氢酶基因在不同开花时期(14,16,18,20d)红花种子中的表达量。【结果】构建的红花种子cDNA文库总克隆数为7.5×106,重组率>95%,插入片段长度均>500bp,全长基因比例达到20%。从红花cDNA文库中共拼接得到contig 543条,singlet 3 444条。delt-12脂肪酸脱氢酶基因序列在成熟红花种子中的表达量较其他时期高。【结论】成功建立了红花种子cDNA文库,克隆了delt-12脂肪酸脱氢酶基因,且该基因表达量在红花种子成熟过程中逐渐升高。

【Objective】The cDNA library of safflower seeds was constructed and safflower fatty acid metabolism pathway genes were analyzed by cDNA library to provide basis for investigation of functional genomes.【Method】The total RNA was extracted from safflower seeds by Trizol method.The cDNA library was constructed by using GeneRacer Kit and CloneMinerⅡ cDNA Library Construction Kit.Recombination rate and insert segments were identified to derive contig and singlet fragments and obtain fatty acid metabolism-related genes.The expressions of fatty acid desaturase genes were measured at different flowering stages（14,16,18 and 20d）by quantitative PCR.【Result】The constructed safflower library contained 7.5×10^6 clones with the recombination rate of greater than 95% and the insert size of greater than500 bp.The ratio of full-length genes reached 20%.The number of contigs obtained was 543 and that ofsinglets was 3 444 after stitching.The expression of delt-12 fatty acid desaturase gene sequence in mature seeds was higher than other periods.【Conclusion】The cDNA library of safflower seed was established successfully.Delt-12 fatty acid desaturase gene was cloned and its expression was the highest in the mature safflower seeds.