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采用同源序列克隆法,结合RACE技术,从三七[Panax notoginseng(Burk.)F.H]根茎总RNA中克隆次生代谢相关差异表达RNase-like贮藏蛋白的编码序列,并进一步以DNA为模板扩增全长基因,获得一条开放阅读框为717 bp的cDNA序列,命名为PMP(GenBank,KC751542),相应基因全长1 074bp。序列及其进化分析表明,该基因包含3个外显子和2个内含子,编码238个氨基酸,相应蛋白质的分子量为27.47 kD,含有核苷酸结合的保守区域,属于RNase-T2超家族成员。三七PMP蛋白与人参RNase-like贮藏蛋白高度同源,序列相似性达95%。实时荧光定量PCR研究表明,三七PMP基因在其根、茎、叶、花等器官中均有表达,且3年生根中表达量最高,暗示该基因可能参与三七皂苷次生代谢调控及其品质形成。
The coding sequence of RNase-like storage protein was differentially expressed in secondary metabolites from total RNA of Panax notoginseng (Burk.) FH] by homologous sequence cloning and RACE, and further amplified with DNA The full-length gene was amplified and a 717 bp open reading frame cDNA sequence was obtained and named PMP (GenBank, KC751542). The corresponding gene was 1 074 bp in length. Sequence and phylogenetic analysis showed that this gene contains 3 exons and 2 introns and encodes 238 amino acids. The corresponding protein has a molecular weight of 27.47 kD and contains conserved regions of nucleotide binding, belonging to the RNase-T2 superfamily member. Panax notoginseng PMP protein and ginseng RNase-like storage protein highly homologous sequence similarity of 95%. Real-time quantitative PCR showed that PMP gene in Panax notoginseng was expressed in roots, stems, leaves, flowers and other organs, and its expression level was the highest in 3-year-old roots, suggesting that this gene may be involved in the regulation of the secondary metabolism of notoginsenoside Quality formation.