重瓣百合LiSEP3基因克隆与表达分析

Cloning and expression analysis of gene LiSEP3 in double lily

【目的】克隆百合LiSEP3基因并确定其在百合不同地上器官及不同花瓣中的表达差异,探讨SEP3基因在重瓣花中的表达模式。【方法】利用RACE方法从重瓣百合品种‘比罗尼卡’中克隆得到LiSEP3基因核苷酸序列,利用SMART软件对其蛋白结构进行分析,以MEGA 5.0软件进行系统进化树分析,用基因枪轰击法进行亚细胞定位分析,采用荧光定量PCR进行不同地上器官及不同花瓣中LiSEP3基因表达差异分析。【结果】LiSEP3基因属于MADS-box家族E类基因,其开放阅读框为729 bp,共编码242个氨基酸;LiSEP3基因编码的蛋白具有典型的MADS结构域、K-box区及2个SEP基序;LiSEP3与同为单子叶植物的六出花的SEP3亲缘关系最近;LiSEP3蛋白定位于细胞核中;LiSEP3基因在花中的相对表达量最高,在茎、叶中几无表达;在1~7轮花瓣中均能检测到LiSEP3表达,且位于最内侧的第7轮花瓣中相对表达量最高。【结论】LiSEP3基因属于MADS-box家族E类基因,主要在花中表达,其中在最内侧花瓣中表达量最高,其表达模式与双子叶植物花中SEP3基因表达模式存在不同。

[ Objetctive] We cloned LiSEP3 from a double lily and analyzed LiSEP3 expression patteru among different aboveground organs and petals, for probing into SEP3 gene expression pattern in the double flower. [ Method ] LiSEP3 was isolated from double lily cv. ＇ Beloniea＇ by RACE, and protein structure of LiSEP3 was predicted by SMART soft- ware. Phylogenetic analysis was performed by MEGA 5.0, subcellular localization of LiSEP3 were detected by particle bombardment. In addition, the expression pattern of LiSEP3 was analyzed among different aboveground organs and petals. [Result] LiSEP3 is a member of E class of MADS-box. The open reading frame of LiSEP3 is 729 bp in length, and encodes a protein of 242 amino acids. Protein structure analysis showed that LiSEP3 has a conserved MADS domain, a K-box region and two SEP motifs. Phylogenetic tree analysis suggested LiSEP3 had the highest similarity with mono- cotyledouous Alstroemeria. Subcellular localization assay revealed LiSEP3 was a nucleus protein. LiSEP3 was abundant in flowers while it couldn＇ t be detected in stems and leaves. LiSEP3 was expressed in petals fi＇om the first whorl to the sev- enth whorl, and had the highest transcription level in the seventh whorl petals. [ Conclusion] LiSEP3 belongs to E class of MADS-box family genes and is specifically expressed in flowers and has a higher expression in seventh whorl than that in other whorls. Moreover, the expression pattern of LiSEP3 in flowers is different from its homologous genes in dicotyle- dons.