芥蓝八氢番茄红素合成酶基因BaPSY1的克隆及原核表达

发布时间：2019-04-01 17:15

【摘要】：本研究根据NCBI公布的白菜、甘蓝等近源物种的同源基因序列设计特异性引物,采用反转录PCR法从‘明丰香菇’芥蓝中克隆到八氢番茄红素合成酶基因BaPSY1,测序结果表明该序列长为1 251 bp,编码氨基酸416个,理论分子量为46.53 k D,等电点为8.83。序列比对发现,芥蓝BaPSY1氨基酸序列与油菜、甘蓝、白菜等十字花科植物相似性较高,达到89%以上,与番茄、甜橙等其他园艺植物的相似性也均超过70%;同源进化树分析表明芥蓝BaPSY1与油菜PSY位于同一分支,亲缘关系最近。此外,芥蓝BaPSY1基因序列中稀有密码子数量较多,所占比例达到11.53%,通过构建原核表达载体pEASY-Blunt E1-BaPSY1,将其转入含稀有密码子的感受态细胞Transetta(DE3)中诱导,最终成功获得了BaPSY1特异性表达蛋白,并且主要以包涵体形式存在。研究结果为揭示芥蓝BaPSY1基因功能提供依据。
[Abstract]:In this study, specific primers were designed according to the homologous gene sequences of Chinese cabbage, cabbage and other close-source species published by NCBI, and the BaPSY1, gene of 8-hydrolycopene synthetase was cloned from 'Mingfeng Lentinula edodes' Brassica oleracea by reverse transcription PCR (RT-PCR). The sequencing results show that the sequence is 1 251 bp, encoding 416amino acids, the theoretical molecular weight is 46.53 KD, and the isoelectric point is 8.83. Sequence comparison showed that the amino acid sequence of kale BaPSY1 was similar to Brassica napus, Brassica oleracea, Chinese cabbage and other cruciferous plants, up to 89%, and over 70% with tomato, orange and other horticultural plants. Phylogenetic tree analysis showed that BaPSY1 and PSY of Brassica napus were located in the same branch and were closely related to each other. In addition, the number of rare codon in the sequence of Chinese kale BaPSY1 gene was more than 11.53%. The prokaryotic expression vector pEASY-Blunt E1 / BaPSY1 was constructed and transferred into competent cell Transetta (DE3) containing rare codon. Finally, the specific expression protein of BaPSY1 was successfully obtained, and it was mainly in the form of inclusion body. The results provide a basis for revealing the function of BaPSY1 gene in Chinese kale.
【作者单位】：四川农业大学园艺学院
【基金】：国家自然科学基金项目(31500247) 四川省教育厅重点项目(14ZA0016)共同资助
【分类号】：Q943.2

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