珠子参β-香树素合成酶基因的克隆和生物信息学分析

发布时间：2018-06-24 11:24

本文选题：珠子参 + β-香树素合成酶　；参考：《生物技术通报》2017年02期

【摘要】：β-香树素合成酶(beta-amyrin synthaseβAS)是定向分流齐墩果烷型皂苷和达玛烷型皂苷的限速酶,对药用植物三萜皂苷的生物合成具有重要的调控作用。基于珠子参转录组数据中βAS转录本序列设计引物,利用RT-PCR方法从珠子参根状茎中克隆得到βAS基因全长c DNA序列,命名为pjβAS。经测序鉴定该基因长度为2 655 bp,包含一个2 286 bp的完整开放阅读框,编码761个氨基酸,在Gen Bank数据库的登录号为KX254566。生物信息学分析显示,其编码蛋白分子量为87.90 k D,理论等电点(p I)5.84,不含跨膜区,为非分泌蛋白,含有38处磷酸化位点,主要于叶绿体和内质网中发挥生理作用;二级结构预测发现其α-螺旋占39.82%、延伸链16.56%、β-转角10.38%、无规则卷曲33.24%,具有DCTAE、QW(QXXXXXW)和MWCRCY3个氧化鲨烯环化酶(OCS)基因家族特征保守基序;pjβAS蛋白与竹节参(AKN23431.1)序列的一致性为100%,系统进化分析中与竹节参(AKN23431.1)、西洋参(AGG09939.1)、柴胡(ABY90140.2)划为同一分支。通过实时荧光定量PCR分析pjβAS在珠子参不同组织中的相对表达量,发现pjβAS基因在花、叶、茎、根状茎中均有表达,在叶中表达量最高,根状茎中表达量最低。
[Abstract]:尾 -vanillin synthase (beta-amyrin synthase 尾 -AS) is a rate-limiting enzyme that distributes Oleanolic saponins and damarane saponins, and plays an important role in regulating the biosynthesis of triterpenoid saponins in medicinal plants. Based on the 尾 -AS transcriptional sequence of Panax japonicus, the full-length cDNA sequence of 尾 -AS gene was cloned from the rhizome of Panax japonicus by RT-PCR and named as pj 尾 ASs. The length of the gene was 2 655 BP, which contained a complete open reading frame of 2 286 BP encoding 761 amino acids. The accession number in GenBank database was KX254566. Bioinformatics analysis showed that the molecular weight of the encoded protein was 87.90 KD, the theoretical isoelectric point (Pi) was 5.84, no transmembrane region, it was a non-secretory protein, and it contained 38 phosphorylation sites, which mainly played a physiological role in chloroplast and endoplasmic reticulum. The secondary structure prediction found that the 伪 -helix occupies 39.82%, the extension chain 16.56, the 尾 -turn 10.38, the irregular crimp 33.240.There is DCTAEEQW (QXXXXXW) and MWCRCY3 family of oxidative squalene cyclase (OCS) genes. The conserved motif pj 尾 -AS is 100 and the sequence of AKN23431.1 is consistent with the sequence of the family of three oxidative squalene cyclase (OCS) genes of DCTAEEQW (QXXXXXW) and MWCRCYs (AKN23431.1). It is classified as the same branch as Radix Bamboo (AKN23431.1), Panax quinquefolium (AGG09939.1) and Bupleurum chinense (ABY90140.2). The relative expression of pj 尾 as in different tissues of Radix Ginseng was analyzed by real-time fluorescence quantitative PCR. It was found that the expression of pj 尾 as gene was found in flowers, leaves, stems and rhizomes, the highest in leaves and the lowest in rhizomes.
【作者单位】：武汉轻工大学生物与制药工程学院;
【基金】：国家自然科学基金(81274023)
【分类号】：S567.53

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