古樟端粒基因CcTBP1和CcPOT1的克隆、亚细胞定位及表达分析  

Cloning,subcellular localization and expression analysis of telomere genes CcTBP1 and CcPOT1 in ancient Cinnamomum camphora

作　　者：刘立盘[1] 杨爱红[1] 刘腾云[1] 钟永达[1] 周华[1] 余发新[1] Liu Lipan;Yang Aihong;Liu Tengyun;Zhong Yongda;Zhou Hua;Yu Faxin(Key Laboratory of Horticultural Plant Genetic Improvement of Jiangxi Province,Institute of Biological Resources,Jiangxi Academy of Sciences,Nanchang 330096,Jiangxi,China)

机构地区：[1]江西省观赏植物遗传改良重点实验室,江西省科学院生物资源研究所,江西南昌330096

出　　处：《北京林业大学学报》2022年第12期1-11,共11页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(32160318);江西省自然科学基金项目(20212BAB205038);江西省科学院包干制项目(2021YSBG22029)。

摘　　要：【目的】端粒基因在植物组织衰老和分化过程中发挥着重要的调控作用。本研究通过对古樟端粒基因CcTBP1和CcPOT1进行克隆与生物信息学分析、亚细胞定位及在古樟及其复幼扦插苗中的表达水平检测,初步揭示端粒基因在古樟复幼扦插不定根发生过程中的功能,为探究古樟复幼的端粒调控机理奠定基础。【方法】本研究以1 200年树龄的古樟为研究材料,提取叶片RNA,利用PCR和同源克隆相结合的方法克隆端粒基因CcTBP1和CcPOT1,并对其序列进行生物信息学分析;通过构建过表达载体进行亚细胞定位分析;以及通过实时荧光定量(qRT-PCR)分析CcTBP1和CcPOT1在古樟及其复幼扦插苗不同组织和不定根发生过程中的表达模式。【结果】(1)克隆获得2个端粒基因,分别命名为CcTBP1和CcPOT1。CcTBP1基因编码序列(CDS)长2 163 bp,编码720个氨基酸;CcPOT1基因CDS序列长1 407 bp,编码468个氨基酸。(2)序列相似性和系统进化树分析结果显示:CcTBP1蛋白与牛樟RWR76720.1序列相似性高达98%,二者亲缘关系最近;CcPOT1蛋白与牛樟RWR96964.1序列相似性高达99%,二者亲缘关系最近。(3)亚细胞定位分析结果显示:CcTBP1蛋白定位在细胞核中,CcPOT1蛋白定位在细胞质和细胞核中。(4)根茎叶qRT-PCR表达分析发现,CcTBP1基因在古樟根中的表达水平最高,CcPOT1基因在复幼扦插苗根中的表达水平最高。不定根发生过程中的表达分析发现:CcTBP1呈下调表达趋势,在古樟不定根中的表达量高于复幼扦插苗;CcPOT1基因呈上调表达趋势,在古樟不定根中的表达量低于复幼扦插苗。【结论】古樟端粒基因CcTBP1和CcPOT1在古樟复幼不定根发生过程中具有重要的调控功能。[Objective] Telomere genes play an important role in the process of plant tissue senescence and differentiation. In this study, the telomere genes CcTBP1 and CcPOT1 of ancient Cinnamomum camphora were cloned and analyzed by bioinformatics, subcellular localization and detection of their expression level in ancient C. camphora and its rejuvenating cuttings, to preliminarily reveal the function of telomere genes in the process of adventitious root formation of ancient C. camphora rejuvenating cuttings, and lay a foundation for exploring the telomere regulation mechanism of ancient C. camphora rejuvenating cuttings.[Method] In this study, leaf RNA was extracted from 1 200 years old ancient C. camphora. The telomere genes CcTBP1 and CcPOT1 were cloned by PCR and homologous cloning, as well the bioinformatics analysis was conducted to their sequences. The over-expression vector was constructed for subcellular localization analysis. The CcTBP1 and CcPOT1 expression level of different tissues and adventitious root formation process was analyzed by qRT-PCR in ancient C. camphora and its rejuvenation cutting seedlings.[Result](1) Two telomere genes were cloned, named CcTBP1 and CcPOT1, respectively. The CDS sequence of CcTBP1 gene was 2 163 bp and encoded 720 amino acids. The CDS sequence of CcPOT1 gene was 1 407 bp and encoded 468 amino acids.(2) The results of sequence similarity and phylogenetic tree analysis showed that the sequence similarity of CcTBP1 protein and Cinnamomum kanehirae RWR76720.1was 98%, and their genetic relationship was the closest;the sequence similarity of CcPOT1 protein and Cinnamomum kanehirae RWR96964.1 was as high as 99%, and their genetic relationship was the closest.(3) Subcellular localization analysis showed that CcTBP1 protein was located in the nucleus, and CcPOT1protein was located in the cytoplasm and nucleus.(4) The qRT-PCR expression analysis in root, stem and leaf showed that the expression level of CcTBP1 gene was highest in root of ancient C. camphora, the expression level of CcPO

关 键 词：古樟 端粒基因 克隆 亚细胞定位 表达分析 

分 类 号：S792.23[农业科学—林木遗传育种]