北美鹅掌楸CCD1基因的克隆与表达分析

发布时间：2018-10-09 09:20

【摘要】：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。
[Abstract]:In this study, 37 EST sequences of CCD gene family were extracted from the transcriptional database of Liriodendron mandshurica and Liriodendron mandshurica, and classified into the CCD gene family. Using one of the EST sequences predicted to be CCD1 as the benchmark primer, a 1 929 bp sequence was cloned from Liriodendron mandshurica by reverse transcription PCR (RT-PCR) and rapid amplification of (RACE) at the c DNA end. The sequence named LtCCD1 gene was cloned from Liriodendron mandshurica (Liriodendron mandshurica). The gene contains an untranslated region sequence of 102 bp 5 '-terminal 177 bp 3' and an open reading frame of 1 650 bp. The cocoding 549aa.LtCCD1 gene is more than 80% similar to the CCD1 gene of many species. The encoded protein possesses the typical domain RPE65. of CCD gene family. Phylogenetic analysis showed that the protein encoded by LtCCD1 gene was closely related to the protein encoded by Anthurium amnicola,Crocus ancyrensis and saffron CCD1 gene. The molecular weight of LtCCD1 protein was 61.96 kD, and the isoelectric point was 6.10, which belonged to acidic protein. Irregular crimp is the main component of the secondary structure of the protein. The online software Target P was used to predict the subcellular localization and combined with the localization information of CCD1 protein in other plants. It was suggested that the protein might be located in the cytoplasm. Tissue specific expression of the gene was analyzed by real-time fluorescence quantitative PCR. It was found that LtCCD1 gene was expressed in eight tissues of Liriodendron mandshurica, including petal, calyx, stamen, pistil, flower bud, leaf bud and stem. The order of expression from large to small is: leaf, calyx, petal, stem, pistil, leaf bud, flower bud, stamen. The results may provide a basis for further analysis of the function of CCD1 gene in Liriodendron mandshurica.
【作者单位】：南京林业大学南方现代林业协同创新中心;
【基金】：国家自然科学基金项目(31470660) 江苏省高校优势学科(PAPD)共同资助
【分类号】：Q943.2;S792.21

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