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目的:克隆得到雷公藤一型4α-甲基氧化酶(Tw SMO1)(Gen Bank KX987126)全长基因,并对其进行序列分析及初步的功能验证。方法:根据雷公藤转录组数据,利用cDNA末端快速扩增(RACE)技术克隆得到雷公藤4α-甲基氧化酶基因(TwSMO1),并利用相关软件对所得核酸序列及其所编码的氨基酸序列进行生物信息学分析。用茉莉酸甲酯(MeJA)诱导雷公藤悬浮细胞,用实时荧光定量(RT-PCR)的方法分析诱导不同时间点后雷公藤悬浮细胞中的TwSMO1基因的相对表达量。结果:克隆所得的TwSMO1基因cDNA全长1505 bp,开放阅读框900 bp,编码299个氨基酸,在线预测其所编码蛋白分子量为34.55 k Da,理论等电点为6.89。多重序列比对结果显示其与其他物种的SMO1氨基酸序列有较高同源性,且包含SMO1基因家族的3个保守域,系统进化树将其归为4α-甲基氧化酶第一家族基因,遂将其命名为TwSMO1。RT-PCR结果表明MeJA诱导后TwSMO1基因在1 h后表达量达到最高,约是对照组的450倍。结论:本研究首次克隆得到雷公藤TwSMO1基因,并对其进行生物信息学分析及MeJA诱导表达分析,为深入研究此基因功能及阐述雷公藤甾醇生物合成途径奠定基础。
OBJECTIVE: To clone and obtain the full-length gene of Tw SMO1 (Gen Bank KX987126), and to perform sequence analysis and preliminary functional verification. Methods: According to the data of Tripterygium wilfordii transcriptome, the gene of TwSMO1 was cloned by rapid amplification of cDNA ends (RACE), and the obtained nucleic acid sequence and its encoded amino acid sequence Bioinformatics Analysis. Tripterygium wilfordii was induced by methyl jasmonate (MeJA), and the relative expression of TwSMO1 gene in suspension cells of Tripterygium wilfordii was analyzed by real-time fluorescence quantitative PCR (RT-PCR). Results: The full length cDNA of TwSMO1 gene was 1505 bp in length and 900 bp in open reading frame (ORF) encoding 299 amino acids. The predicted protein molecular weight was 34.55 kDa and the theoretical isoelectric point was 6.89. The results of multiple sequence alignment showed that it shared high homology with the SMO1 amino acid sequence of other species and contained three conserved domains of SMO1 gene family. The phylogenetic tree was classified as the first family of 4α-methyl oxidase genes, It is named TwSMO1. The results of RT-PCR showed that the expression of TwSMO1 gene reached the highest level at 1 h after MeJA induction, which was about 450 times of that of control group. Conclusion: TwSMO1 gene was first cloned and analyzed by bioinformatics analysis and MeJA-induced expression analysis in order to further study the function of this gene and elucidate the pathways involved in the sterol biosynthesis.