长柄石杉L-赖氨酸脱羧酶基因克隆及序列分析

目的在对长柄石杉Huperzia serrata、闽浙马尾杉Phlegariurus minchegensis、华南马尾杉Phlegariurus austrosinicus和有柄马尾杉Phlegariurus petiolatus 4种石杉科植物的L-赖氨酸脱羧酶(L-lysine decarboxylase,LDC)基因编码区进行克隆的基础上,对长柄石杉的LDC基因全长序列进行分析并预测其蛋白结构。方法采用RT-PCR技术,以石杉科植物叶片提取的总RNA为模板克隆得到LDC基因编码区序列,通过BLAST比对和MEGA5.0软件对克隆的序列进行分析。采用RACE技术获得石杉科广布种长柄石杉的LDC基因全长序列,并对其蛋白的二级结构及三维结构进行预测分析。结果克隆的4种石杉科植物LDC基因序列与数据库中被注释为编码LDC的基因序列保持高度的相似性,长柄石杉LDC蛋白与NCBI中的蛇足石杉LDC氨基酸序列相似度很高,与蕨类植物江南卷柏的同源性也较高。长柄石杉LDC基因编码区全长为1 266 bp,编码403个氨基酸,Gen Bank登录号KF040056。结论克隆得到4种石杉科植物的LDC基因,分析了长柄石杉LDC基因的全长序列并预测其蛋白结构,为进一步阐明石杉科植物石杉碱甲生物合成途径奠定基础。

Cloning and sequence analysis of L-lysine decarboxylase gene in Huperzia serrata var. longipetiolata

Objective To obtain and analyze the full-length L-lysine decarboxylase (LDC) gene sequence of Huperzia serrata var. longipetiolata and predictively analyze its protein structure on the basis of cloning the coding region of LDC gene from four species of Huperziaceae. The species are Huperzia serrata var. longipetiolata, Phlegariurus minchegensis, Phlegariurus austrosinicus, and Phlegariurus petiolatus. Methods The LDC coding region sequences were cloned by RT-PCR strategy with the template of total RNA extracted from the leaves. Then the sequences were analyzed by means of BLAST and MEGA 5.0. The full-length of LDC gene sequence of H. serrata var. longipetiolata was obtained by RACE technology. And then the secondary structure and three-dimensional structure of LDC protein were predictively analyzed. Results The coding region sequences were highly similar to the lysine decarboxylase in the database. And the encoding protein of H. serrata var. longipetiolata was highly similar to the amino acid sequences of H. serrata in NCBI, and with high homology to Selaginella moellendorffii. The full-length LDC gene sequence of H. serrata var. longipetiolata contained 1 266 bp open reading frame and encodes a predicted protein of 403 amino acids. The GenBank accession number for this gene is KF040056. Conclusion The LDC genes of the four species of Huperziaceae are cloned in this study. The full-length LDC gene sequence of H. serrata var. longipetiolata is obtained and analyzed, and its protein structure is predictively analyzed. The result will provide a foundation for exploring the mechanism of huperzine A biosynthesis in the plants of Huperziaceae.