银杏叶绿体petD基因的克隆与表达

Cloning and Expression of A Chloroplast petD Gene from Ginkgo biloba L.

根据黑松、云杉、菠菜与玉米叶绿体petD基因序列设计引物,以银杏叶绿体基因组DNA为模板,PCR扩增克隆了银杏叶绿体petD基因(GenBank登录号为DQ923066,命名为GbpetD)的序列。序列分析显示,GbpetD基因组DNA序列编码区长1243bp,含1个内含子和2个外显子,其外显子序列编码177个氨基酸。相似性比对显示,该基因编码区序列与云杉、台东苏铁、黑松、莴苣、木薯、北美落叶松的petD基因核苷酸同源性为84%～99%,氨基酸序列同源性为85%～93%。系统进化树分析结果表明GbpetD蛋白质与黑松、北美落叶松、云杉、苏铁等裸子植物的petD蛋白质聚类关系最近。半定量RT-PCR分析表明,GbpetD基因在银杏叶和茎中表达,在叶中表达量最大。

A chloroplast petD gene, named GbpetD, was cloned from Ginkgo biloba using a pair of primers, which were designed according to the homologous sequences in Pinus thunbergii, Picea abies, Spinacia oleracea, and Zea mays. The genomic DNA of GbpetD had 1 243 bp coding sequence, including one intron and two exons, which encoding a 177-amino-acid protein. The Blast results showed that the homology of the GbpetD nucleotide sequence with petD genes from P. abies, Cycas taitungensis, P. thunbergii, Lactuca sativa, Manihot esculenta, and Larix occidentalis was from 84% to 99%, and the homology of amino acid sequences was from 85% to 93%. Phylogenetic tree analysis indicated that the GbpetD protein had the closest relationship with petD proteins from gymnosperm plants. The expression analysis with semi-quantitative RT-PCR revealed that GbpetD gene expressed in leaves and young stems, and the highest level was detected in leaves.