摘要
为了建立一种检测独一味mRNA表达水平的SYBR Green Ⅰ荧光定量Real-Time PCR方法.根据独一味查尔酮合成酶（LrCHS）和苯丙氨酸解氨酶（LrPAL）基因序列设计特异性引物,运用Real-Time RT-PCR方法建立独一味基因的荧光定量标准曲线,进一步研究独一味CHS和PAL基因在不同组织中的表达.结果显示,扩增曲线在1×10^5～1×10^11 copies/μL范围内有很好的线性关系,扩增相关系数在0.995以上.熔解曲线分析表明,产物为特异单峰,无引物二聚体,具有较高的特异性和灵敏度.独一味CHS基因在不同组织中表达量顺序为花＞叶＞叶柄＞根＞茎,PAL基因表达量顺序为叶柄＞叶＞花＞根＞茎.建立的检测方法能够成功用于独一味CHS和PAL基因表达的检测,为研究独一味在mRNA水平的定量分析提供技术平台.
This study developed a real-time reverse-transcription polymerase chain reaction （RT-PCR） method based on SYBR Green I fluorescent for detection of Lamiophlomis rotate （Benth.） gene mRNA. According to the gene sequence in the conservative regions of chaleone synthase （LrCHS） and phenylalanine ammonia-lyase （LrPAL） in L. rotate, the specific primers were designed with LrCHS and LrPAL to construct real-time RT-PCR assay. The standard curves showed good linear relationships with the correlation eoffieient abave 0.995 in the range of 1×10^5～1×10^11 copies/μL.The melting curve analysis showed that the product was specific to a single peak,and no primer-dimers ,with high specificity and sensitivity. The expression level of CHS gene in different organization of L. rotate showed flowers〉leaves〉petioles〉roots〉stems trend, and the expression level of PAL gene showed petloles〉leaves〉flowers〉roots〉stems trend. This assay could successfully provide a technical platform to research L. rotate genes at the mRNA level in the quantitative analysis.
出处
《广东农业科学》
CAS
CSCD
北大核心
2014年第12期133-137,I0002,共6页
Guangdong Agricultural Sciences
基金
教育部春晖计划项目(Z2010076)
