山韭蒜氨酸酶基因（AsALy1）全长cDNA的克隆及表达分析

发布时间：2019-02-22 12:05

【摘要】：山韭为百合科多年生的野生葱属植物,是中国传统药材,其有效的药用成分具有抗病原微生物、抗氧化、抗肿瘤、降血压、降血脂、降血糖、抗血小板聚集以及护肝等生理学功能,而这药用成分主要是蒜素。蒜素是通过蒜氨酸酶与蒜氨酸发生反应而产生的有机硫化物之一,因此克隆及分析蒜氨酸酶基因,不仅为该酶的进一步研究提供一定的理论依据,同时,为蒜素在医药领域的工业生产应用奠定了基础。1本研究以山韭为实验材料,进行山韭蒜氨酸酶(暂命名为AsALy1)基因cDNA序列的克隆、表达量分析以及生物信息学分析。具体研究方法:利用RT-PCR技术克隆AsALy1基因和山韭actin基因的保守区序列;利用RACE技术克隆As ALy1基因的3'/5'端未知序列和ORF序列,并通过电子拼接得到AsALy1基因的全长cDNA序列;通过实时荧光定量PCR技术对AsALy1基因在山韭不同组织中表达量进行分析;运用生物信息学分析As ALy1基因的氨基酸序列。研究结果:克隆出的AsALy1基因保守区片段的长度为862 bp;利用RACE技术获得了长度为1655 bp的AsALy1基因的全长cDNA;利用生物信息软件分析表明,其开放阅读框(ORF)长度为1440 bp,编码479个氨基酸的蛋白质,相对分子质量为54950.82 Da,理论等电点为8.75;预测As ALy1蛋白在13 32位氨基酸之间有个跨膜区;二级结构主要由α螺旋、随机卷曲和β折叠链组成;预测的蒜氨酸酶具有天冬氨酸氨基转移酶超家族结构域、C-末端的催化结构域、类表皮生长因子结构域以及天冬氨酸/蛋氨酸/酪氨酸转氨酶结构域;预测该蛋白质具有丝氨酸、苏氨酸、酪氨酸三种磷酸化位点和N-糖基化位点。克隆出的山韭actin基因保守区片段的长度为386 bp;经过实时荧光定量PCR技术得出AsALy1基因在茎中的表达量分别是花、叶和种子的0.7倍、36倍和157倍,而AsALy1基因在根中几乎不表达,表明该酶是鳞茎蒜氨酸酶。
[Abstract]:Chinese chive is a perennial wild Allium plant of Liliaceae. It is a traditional Chinese medicine. Its effective medicinal ingredients are anti-pathogenic microorganism, anti-oxidation, anti-tumor, lowering blood pressure, lowering blood lipid, lowering blood sugar. Antiplatelet aggregation and liver protection and other physiological functions, and this medicinal ingredient is mainly allicin. Alliin is one of the organic sulfides produced by the reaction of alliinase with alliinic acid. Therefore, cloning and analyzing alliinase gene not only provides a theoretical basis for further study of alliinase, but also provides a theoretical basis for further study of alliinase. In this study, the cDNA sequence of alliinase gene (tentatively named AsALy1) was cloned, expressed and bioinformatics were analyzed. Methods: the conserved regions of AsALy1 gene and actin gene were cloned by RT-PCR. The unknown and ORF sequences of As ALy1 gene were cloned by RACE technique, and the full-length cDNA sequence of AsALy1 gene was obtained by electronic splicing, and the expression of AsALy1 gene in different tissues was analyzed by real-time fluorescence quantitative PCR technique. The amino acid sequence of As ALy1 gene was analyzed by bioinformatics. Results: the length of the conserved region of the cloned AsALy1 gene was 862 bp;. The full-length cDNA; of the AsALy1 gene with the length of 1655 bp was obtained by RACE technique. The analysis of bioinformatics software showed that the open reading frame (ORF) length was 1440 bp, and the relative molecular weight was 54950.82 Da, theoretical isoelectric point was 8.75; It is predicted that the As ALy1 protein has a transmembrane region between the amino acids of 13 and 32, and the secondary structure is mainly composed of 伪 helix, random crimp and 尾 -fold chain. The predicted alliinase has superfamily domain of aspartate aminotransferase, catalytic domain of C-terminal, epidermal growth factor domain and aspartate / methionine / tyrosine transaminase domain. It was predicted that the protein had three phosphorylation sites, serine, threonine and tyrosine, and N-glycosylation sites. The length of the conserved fragment of actin gene was 386 bp;. The results of real-time fluorescence quantitative PCR showed that the expression of AsALy1 gene in stem was 0.7 times, 36 times and 157 times as much as that in flower, leaf and seed, while AsALy1 gene was almost not expressed in root, which indicated that AsALy1 gene was alliinase in bulb.
【学位授予单位】：内蒙古师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S567.239

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