[木奈]肉桂酸-4-羟化酶基因及其启动子克隆与表达分析

Cloning and Expression Analysis of Cinnamate 4-hydroxylase Gene from Prunus salicina and Isolation of Its Promoter

以不同发育时期的果实为试材,采用两步法逆转录与抑制PCR技术相结合的方法构建均一化全长cDNA文库,筛选出一条编码肉桂酸-4-羟化酶的全长cDNA(命名为PsC4 H);采用APA-Walking技术,分离获得该基因的启动子序列;采用实时荧光定量PCR技术,检测该基因在果实不同发育时期的表达动态。结果表明:PsC4 H基因全长为1 772bp,ORF(Open Reading Frame)为1 515bp,编码504个氨基酸,相对分子量为145 719.6,等电点为4.94,经序列同源性分析发现,PsC4 H氨基酸序列与李属果树高度同源;经PlantCare软件预测,PsC4 H基因的启动子除具有TATA/CAAT-box外,还含有G-box、HSE等特异作用元件;实时荧光定量结果显示,PsC4 H在果实整个生长发育过程中呈下调-上调的表达趋势,其中在成熟果中表达量最高。

Taking the fruits of different development stages of Prunus salicinaas materials,a normalized full-length cDNA library was built combining with two step method of reverse transcription and inhibit PCR technology,a full lenghth cDNA encoded Cinnamate 4-hydroxylase（C4H）（named PsC4 H）was separated;the promoter was isolated by genome walking technology,changes of expression of the gene was detected by Real-time PCR at different development stages fruits.The results showed that the full-lenghth PsC4 H was 1 772 bp with an open reading frame 1 515 bp and encoding504 amino acids with a calculated molecular weight of 145 719.6and theoretical pI of 4.94.Sequence homology analysis indicated that the amino acid sequence of PsC4 Hexhibit high homology to prunus plants.By PlantCare Software predict that in addition to TATA/CAAT-box,still contained some specific regulatory elements such as G-box,HSE and etc.Real time PCR experiment showed that the expression of PsC4 H gene was down-up regulation through the entire developmental periods and was the highest at mature fruit stage.