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。
β-amyrin synthase ( βAS ), the rate-limiting enzyme saponin of directional shunt oleanane and dammarane types, plays an important role in biosynthesis of triterpenoid saponin in medical plants. The primers were designed based on the transcript sequence of βAS from the transcriptome dataset of P. japonicus var. major. And the full-length cDNA sequence of βAS was cloned from the rhizome of P, japonicus var. major by utilizing RT-PCR method, named as pjβAS. The length of this gene was 2 655 base pairs containing a complete open reading frame of 2 286 base pairs and encoding 761 amino acids. The accession number in GenBank is KX254566. The bioinformatics analysis showed that the putative molecular weight and theory isoelectric point of protein encoded by pjflAS were 87.90 kD and 5.84, respectively. The protein contained no transmembrane domain, was a non-secretory protein with 38 phosphorylation sites, it played a physiological role in chloroplast and endoplasmic reticulnm. The secondary structure of the protein indicated that or-helix accounted for 59.82, extended strand for 16.56%, β-turn for 10.38%, and random coil for 33.24%. Meanwhile, three feature conserved motifs of oxido squalene cyclase ( OSC ) were found in pjβAS, i.e., DCTAE, QW ( Qxxxxxw ), and MWCRCY. The results of the sequence homology comparison concluded that the protein encoded by piCAS was in 100% similarity with Panaxjaponicus C. A. Mey ( AKN23431.1 ), and pjI-AS gene was classified in the same branch with Panax japonicus C. A. Mey ( AKN23431.1 ), Panax quinquefolius ( AGG09939.1 ), and Bupleurum chinense DC ( ABY90140.2 ) in phylogenetic analysis. Real-time fluorescence quantitative PCR was used to analyze the relative expression quantity of pjβAS gene in different tissues of P. japonicus var. major, the results showed that there were different expression trends in flowers, leaves, stems and rhizomes, with the highest in leaves but the lowest in rhizomes.
出处
《生物技术通报》
CAS
CSCD
北大核心
2017年第2期109-117,共9页
Biotechnology Bulletin
基金
国家自然科学基金(81274023)
关键词
珠子参
β-香树素合成酶
三萜皂苷
基因克隆
生物系信息学分析
Panaxjaponicus var. major
β-amyrin synthase
triterpenoid saponin
gene cloning
bioinformatics analysis