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本研究以北美鹅掌楸为试验材料,从北美鹅掌楸和鹅掌楸转录组数据库中挖掘出37条CCD基因家族的EST序列,并将这些序列归类于CCD基因家族中。以其中一条预测为CCD1的EST序列为基准设计引物,通过反转录PCR(RT-PCR)与c DNA末端快速扩增(RACE)技术从北美鹅掌楸中克隆得到一条全长为1 929 bp的序列,命名为LtCCD1基因。该基因包含102 bp 5’端、177 bp 3’端非翻译区序列和1 650 bp的开放阅读框,共编码549aa。LtCCD1基因与多个物种CCD1基因相似度大于80%,其编码的蛋白质拥有CCD基因家族典型的结构域RPE65。系统发育分析发现,LtCCD1基因编码的蛋白质与Anthurium amnicola、Crocus ancyrensis和番红花的CCD1基因编码的蛋白质亲缘关系较近。LtCCD1蛋白质分子量为61.96 kD,等电点为6.10,属酸性蛋白;无规则卷曲是该蛋白质二级结构的主要成分。利用在线软件Target P进行亚细胞定位预测并结合CCD1蛋白在其它植物中的定位信息,推测该蛋白可能位于细胞质内。使用实时荧光定量PCR技术对该基因进行组织特异性表达分析,发现LtCCD1基因在北美鹅掌楸的花瓣、花萼、雄蕊、雌蕊、花芽、叶、叶芽、茎8个组织中均有表达,表达量从大到小的顺序依次为:叶、花萼、花瓣、茎、雌蕊、叶芽、花芽、雄蕊。该研究结果可为进一步分析北美鹅掌楸中CCD1基因的功能奠定基础。
In this study, we used the Liriodendron tulipifera as test material to excise the EST sequences of 37 CCD gene families from the transcription database of Liriodendron tulip and Liriodendron, and classify these sequences into the CCD gene family. One of the primers was designed based on the EST sequence of CCD1. A total length of 1 929 bp was obtained from the roots of Liriodendron tulipifera by RT-PCR and rapid cDNA amplification (RACE) The sequence, named LtCCD1 gene. The gene contains 102 bp 5 ’end, 177 bp 3’ untranslated region and 1 650 bp open reading frame, encoding a total of 549aa. The similarity between LtCCD1 gene and multiple species CCD1 gene is greater than 80%, and the encoded protein has the typical domain RPE65 of CCD gene family. Phylogenetic analysis revealed that the protein encoded by LtCCD1 gene has a close relationship with the protein encoded by the CCD1 gene of Anthurium amnicola, Crocus ancyrensis and saffron. LtCCD1 protein molecular weight of 61.96 kD, isoelectric point of 6.10, is an acidic protein; random curl is the main component of the secondary structure of the protein. Using online software Target P for subcellular localization prediction and combining with the localization information of CCD1 protein in other plants, it is presumed that the protein may be located in the cytoplasm. Tissue-specific expression analysis of this gene using real-time fluorescence quantitative PCR revealed that LtCCD1 gene was expressed in 8 tissues of petals, calyces, stamens, pistils, flower buds, leaves, leaf buds and stems of Liriodendron livre. The expression level From big to small in descending order: leaves, calyx, petals, stems, pistils, leaf buds, flower buds, stamens. The results of this study may provide a basis for further analysis of CCD1 gene function in Liriodendron tulipifera.