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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。
According to the petD gene sequences of chloroplasts from Pinus thunbergii, Spruce, Spinach and maize, the petD gene of Ginkgo biloba (GenBank accession number DQ923066, named GbpetD) was cloned by PCR using genomic DNA from chloroplast of Ginkgo biloba as template. Sequence analysis showed that the coding region of GbpetD genomic DNA was 1243bp in length, containing 1 intron and 2 exons, and its exon sequence encoded 177 amino acids. The similarity alignment showed that the nucleotide sequence of the petD gene was 84% -99% homologous to the petD gene of Spruce, Taitung cycad, Pinus thunbergii, Lettuce, Cassava, and North American larch. The amino acid sequence homology 85% ~ 93%. The results of phylogenetic tree analysis showed that the petb protein clustering relationship between GbpetD protein and gymnosperm such as Pinus thunbergii, Larch, Spruce and Cycas was the closest. Semi-quantitative RT-PCR analysis showed that GbpetD gene was expressed in leaves and stems of Ginkgo biloba, and its expression was the highest in leaves.