【作者】 李志强；

【导师】 涂知明； 何光源；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 类胡萝卜素是自然界存在的一类由异戊二烯为结构单位组成的化合物的总称,其按照是否含氧可分为胡萝卜素和叶黄素两大类。类胡萝卜素广泛存在于部分细菌、真菌和植物中。很多类萝卜素有着独特的生理功能,对人体健康起到了非常重要的作用。在小麦中构建类胡萝卜素生物合成途径,使类胡萝卜素积累,从而提高小麦的营养价值。本实验根据发表的小麦八氢番茄红素合成酶基因(PSY1)序列设计特异性引物,以小麦叶片总RNA为模板,用RT-PCR方法扩增出1400 bp大小的片段,并将其连接到pMD18-T载体上转化进大肠杆菌感受态细胞。测序结果显示,克隆的基因序列全长1436 bp,包含一个1287 bp的完整的开放阅读框,编码的蛋白质由428个氨基酸构成。经过生物信息学分析,发现此基因与其它物种的PSY基因中的PSY1同源性更高,确认是小麦中的PSY1基因。将得到的小麦PSY1基因片段连接到原核表达载体pET-32a上,构建重组表达载体,转化大肠杆菌感受态细胞,用IPTG进行诱导表达。经SDS-PAGE分析,诱导的重组菌株与未经诱导的对照组相比,在相对分子量约47kD附近有明显的蛋白质表达条带。本实验成功地对小麦八氢番茄红素合成酶基因进行了克隆和表达,为小麦八氢番茄红素合成酶基因进一步的深入研究奠定了基础。更多还原

【Abstract】 Carotenoids, which can be divided into carotenes and its oxidative derivatives (xanthophylls), are one large group of natural products that are mostly synthesized from isoprene units. They can be biosynthesized in many kinds of bacterium, fungi and plants and many of them are closely related to human health due to their special physiological functions. The nutritional value will be evidently promoted by improving carotenoid contents through building carotenoid synthasizing pathway in wheat.A pair of RT-PCR primers were designed and synthesized based on the published gene sequence of wheat PSY1. About 1400 bp target DNA sequence were amplified by reverse transcription polymerase chain reaction depending on the template of total RNA isolated from wheat leaves, and cloned to pMD18-T simple vector, transformed into E.coli competent cells. The result showed that the length of the acquired clone was 1436 bp, containing a complete Open Reading Frame of 1287 bp which encode a protein of 428 amino acids. We found that the clone had high homology with other species’s PSY1 gene through cladogram analysis,and confirmed it was the wheat PSY1 .By the technology of DNA recombination, the wheat PSY1 clone was subcloned to BamHI/SalI sites of the expression plasmid pET-32a vector. The recombined plasmid, named pET-32a-PSY, was transformed into E.coli BL21 competent cells and induced with IPTG. SDS-PAGE illuminated that about 47 kD protein was expressed in the induced recombinant E.coli BL21, with nothing in the control group.In conclusion, we successfully cloned wheat PSY1 and constructed the recombinant pET-32a-PSY which was expressed in prokaryotic E.coli BL21 cells. All underlied the future research on wheat phytoene synthase gene.更多还原