红花生育酚环化酶基因克隆及表达分析.docVIP

红花生育酚环化酶基因克隆及表达分析 　　[摘要]生育酚环化酶（tocopherol cyclase， TC）是植物Vit E生物合成途径的关键酶之一。该研究根据红花转录组数据，利用RT-PCR（reverse transcription-PCR）方法从药用植物红花种子中克隆得到生育酚环化酶基因序列，命名为CtTC， 对其进行生物信息学分析，结果表明该基因开放阅读框（ORF）为1 524 bp，编码507个氨基酸，相对分子质量为62.9 kDa，等电点（theoretical pI）为5.01。跨膜结构分析表明TC存在跨膜结构。蛋白糖基化位点分析表明TC蛋白含有1个丝氨酸糖基化位点以及1个苏氨酸糖基化位点。亚细胞定位表明其定位于叶绿体中。利用荧光定量PCR检测红花不同发育时期种子以及干旱胁迫不同时间的红花叶片中TC基因的表达量，结果表明TC基因在红花的开花后50 d的种子中表达量最高，干旱胁迫6 d时TC基因的表达量达到最高值，随后下降，为TC基因在红花Vit E合成以及抵抗逆境胁迫中的功能研究奠定基础。 　　[关键词]红花；生育酚环化酶；克隆；表达分析 　　[Abstract]The tocopherol cyclase was one of the key enzymes in plant vitamin E biosynthesis pathway. According to the study of Carthamus tinctorius transcriptome data，the Tocopherol cyclase gene was obtained using RT-PCR techniques and named CtTC . Bioinformatics analysis showed theopen reading frame （ORF）of CtTC was 1 524 bp. The putative protein contained 507 amino acids with a predicted molecular mass of 62.9 kDa and theoretically isoelectric point was 5.01.Signal peptide analysis showed that it was a non secretory protein， and there was no signal peptide. The subcellular localization showed that the CtTC protein was located in the chloroplast. The expression of CtTC gene in safflower seeds at different development stages was determined by quantitative real-time PCR， it was found that the highest expression level of CtTC gene was detected in 50 DAF.Quantitative RT-PCR analysis suggested that expression of CtTC is induced and strengthened by drought stresses. This research provided a candidate gene for metabolic engineering of vitamin E and resisting stress. 　　[Key words]Carthamus tinctorius； tocopherol cyclase； clone； expression analysis 　　doi：10.4268/cjcm 　　红花Carthamus tinctorius L， 又称草红花或刺红花， 为一年生或两年生菊科植物[1]， 在中国大部分地方均有栽培， 主要分布于河南、 四川、 新疆、 浙江等地。红花籽，又称“白平子”，是传统中药红花的种子，李时珍在《本草纲目》中称其“功能与花同”[2]。红花籽中富含Vit E、不饱和脂肪酸等，被广泛应用于食品、保健品等领域。 　　红花中有效成分有黄酮类物质、不饱和脂肪酸、Vit E等，红花有效成分种类以及药理作用的研究已有大量报道[3-4]。近年来，随着新一代测序技术的广泛应用，人们从基因组、转录组角度对红花进行研究。刘秀明等[5]应用454测序技术对红花C. tinctorius初花期和盛花期2个样本进行