三七RNase-like基因的克隆及表达分析

Isolation and Expression Analysis of RNase-like Gene from Panax notoginseng

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。

Combining homology cloning approaches with RACE （rapid amplification of cDNA ends） techniques, the coding sequence of RNase-like major storage protein with differential expression was cloned from the total RNA of roots and stems of Panax notoginseng, then the full gene sequence was amplified from the total DNA. As a result, a cDNA sequence containing a 717 bp ORF （open reading frame） was cloned and named as PMP （GenBank, KC751542 ）, together with a full-length DNA sequence of 1 074 bp. Analysis of sequence and its phylogenetic tree showed that PMP gene consisted of 2 introns and 3 exons, encoding a protein of 238 amino acids. The deduced protein, with a predicted molecular mass of 27.47 kD, contained two conserved domains of RNases, which belonged to the RNase-T2 superfamily member. The sequence showed 95% identity with that of RNase-like major storage protein in Panax ginseng. Real-time quantitative PCR showed that the expression level of PMP in 3-year-old root was higher than the other organs. The expression pattern of PMP showed notable correlation with that of main active components in P. notoginseng, which suggested that it might be involved in the regulation of secondary metabolism of notoginsenoside and the quality formation.