北美鹅掌楸CCD1基因的克隆与表达分析  

Cloning and Expression Analysis of CCD1 Gene in Liriodendron tulipifera

作　　者：成彦丽 仲维平[1] 郝自远 李火根[1] 

机构地区：[1]南京林业大学南方现代林业协同创新中心,南京210037

出　　处：《分子植物育种》2017年第6期2139-2146,共8页Molecular Plant Breeding

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

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。In this paper, 37 EST sequences were mined from the transcriptome data of Liriodendron tulipifera/Liriodendron chinense and classified into CCD gene family. The CCD1 gene was cloned from L. tulipifera using the reverse transcription PCR（RT-PCR） and the rapid amplification of c DNA ends（RACE） methods and named LtCCD1. The full-length 1 929 bp sequence of LtCCD1 gene contains 177 bp 3＇RACE fragment, 102 bp 5＇ RACE fragment and 1 650 bp open reading frame（ORF）, encoding 549 aa. The sequence of LtCCD1 showed high similarity with other plants. LtCCD1 protein contained typical RPE65 domain structure of CCD gene family and especially closest with Anthurium amnicola, Crocus ancyrensis and Crocus sativus according to the results of phylogenetic analysis. The molecular weight of predicted LtCCD1 acidic protein was 61.96 kD with a theoretical isoelectric point of 6.10. Random coil is the major components of secondary structure of this protein. The LtCCD1 protein was predicted locating in cytoplasm. Expression profile by Real-time PCR showed that the gene was expressed in all eight tissues studied, while the expression levels varied significantly among eight tissues. The tendency of expression level of LtCCD1 gene was, from high to low, leaf, calyx, petal, stem, pistil, leaf bud, flower bud, and stamen respectively. This result will provide foundation for future studies on CCD1 gene function in L. tulipifera.

关 键 词：北美鹅掌楸 CCD1基因 克隆 序列分析 表达 

分 类 号：Q943.2[生物学—植物学] S792.21[农业科学—林木遗传育种]