广西师范大学学报（自然科学版） ›› 2025, Vol. 43 ›› Issue (4): 213-223.doi: 10.16088/j.issn.1001-6600.2024110104

• 生态环境科学研究 • 上一篇    下一篇

杉木ClHSP70基因的克隆及表达模式分析

郭胜周1,2,3, 许祖元1,2,3, 刘荣林1,2,3, 林秦民4, 曹光球1,2,3, 曹世江1,2,3*   

  1. 1.福建农林大学 林学院, 福建 福州 350002;
    2.国家林业和草原局杉木工程技术研究中心(福建农林大学), 福建 福州 350002;
    3.林木逆境生理生态及分子生物学福建省高校重点实验室(福建农林大学), 福建 福州 350002;
    4.福建农林大学 生命科学学院, 福建 福州 350002
  • 收稿日期:2024-11-01 修回日期:2024-12-02 出版日期:2025-07-05 发布日期:2025-07-14
  • 通讯作者: 曹世江(1984—),男,吉林长春人,福建农林大学副教授,博士。E-mail: csjiang1123@126.com
  • 基金资助:
    “十四五”国家重点研发计划项目(2021YFD2201302)

Cloning and Expression Pattern Analysis of ClHSP70 Gene in Chinese fir

GUO Shengzhou1,2,3, XU Zuyuan1,2,3, LIU Ronglin1,2,3, LIN Qinmin4, CAO Guangqiu1,2,3, CAO Shijiang1,2,3*   

  1. 1. College of Forestry, Fujian Agriculture and Forestry University, Fuzhou Fujian 350002, China;
    2. Chinese Fir Engineering Research Center of National Forestry and Grassland Administration (Fujian Agriculture and Forestry University), Fuzhou Fujian 350002, China;
    3. University Key Laboratory of Forest Stress Physiology, Ecology and Molecular Biology of Fujian Province (Fujian Agriculture and Forestry University), Fuzhou Fujian 350002, China;
    4. College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou Fujian 350002, China
  • Received:2024-11-01 Revised:2024-12-02 Online:2025-07-05 Published:2025-07-14

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摘要： 本研究通过克隆、生物信息学和表达分析方法对杉木热激蛋白70(heat shock protein 70,HSP70)基因进行研究,为深入理解杉木HSP70基因的功能和杉木遗传改良以及其可持续栽培提供科学依据。本研究选用杉木优良无性系“洋061”一年生苗作为试验材料,利用逆转录聚合酶链式反应(RT-PCR)克隆得到ClHSP70基因。运用Expasy软件等在线软件预测并分析ClHSP70蛋白的理化性质、跨膜螺旋域、信号肽、二级结构和三级结构;运用Cell-PLoc 2.0在线软件预测蛋白的亚细胞定位;运用Mega 11软件构建系统发育树;克隆ClHSP70基因并构建到pCAMBIA35s-EGFP载体中,分析ClHSP70蛋白的亚细胞定位;并利用实时荧光定量PCR(quantitative real-time PCR)对其表达水平进行分析。克隆得到的ClHSP70基因编码670个氨基酸,ClHSP70蛋白的分子式为C3297H5306N940O1008S26,具有不稳定性,不含信号肽和跨膜区域,预测定位于细胞质。系统进化分析表明,杉木ClHSP70与欧榛Corylus avellana亲缘关系更为密切。亚细胞定位实验结果显示,ClHSP70蛋白定位于细胞核;qRT-PCR表达分析,ClHSP70基因在叶片中的相对表达量最高。ClHSP70基因在高温条件下6 h后相对表达量达到峰值,干旱处理12 h其表达量达到最大值,即ClHSP70基因受高温和干旱胁迫诱导上调表达。杉木ClHSP70基因的成功克隆与序列分析,揭示了其在杉木不同组织中的表达及对高温与干旱胁迫的响应,并为杉木抗逆性育种提供重要的理论基础。

关键词: 杉木, ClHSP70基因, 基因克隆, 亚细胞定位, 高温与干旱胁迫

Abstract: In this study, the Heat Shock Protein 70 (HSP70) gene of cedar was investigated by cloning, bioinformatics and expression analysis methods to provide a scientific basis for the in-depth understanding of the function of the HSP70 gene of cedar, as well as the genetic improvement and sustainable cultivation of cedar. The annual seedlings of the excellent Chinese fir asexual line “Yang 061” were selected as test materials, and the ClHSP70 gene was cloned by reverse transcription polymerase chain reaction (RT-PCR). The physicochemical properties, transmembrane helical domain, signal peptide, secondary structure and tertiary structure of ClHSP70 protein were predicted and analysed using Expasy software and other online software; the subcellular localization of the protein was predicted using Cell-PLoc 2.0 online software; Mega 11 software was used to construct a phylogenetic tree; the ClHSP70 gene was cloned and constructed into pCAMBIA35s-EGFP vector for analysis of ClHSP70 gene. EGFP vector to analyse the subcellular localisation of the ClHSP70 protein; and its expression level was analysed using Quantitative Real-time PCR (QRP). The cloned ClHSP70 gene encoded 670 amino acids, and the molecular formula of ClHSP70 protein was C3297H5306N904O1008S26, which was unstable, did not contain signal peptide and transmembrane region, and was predicted to be localised in the cytoplasm. Phylogenetic analysis showed that Chinese fir ClHSP70 was more closely related to hazelnut (Corylus avellana). The results of subcellular localisation experiments showed that ClHSP70 protein was localised in the nucleus. qRT-PCR expression analysis showed that the relative expression of ClHSP70 gene was the highest in the leaves, and the relative expression of ClHSP70 gene reached the peak after 6 h of high temperature, and the expression of ClHSP70 gene reached the maximum after 12 h of drought treatment, and the expression of ClHSP70 gene was up-regulated by the high temperature and drought stress induced. The ClHSP70 gene was up-regulated by high temperature and drought stress. The successful cloning and sequence analysis of the ClHSP70 gene in cedar revealed its expression in different tissues of cedar and its response to high temperature and drought, and provided an important theoretical basis for the breeding of cedar resistance.

Key words: Chinese fir, ClHSP70 gene, gene cloning, subcellular localization, high temperature and drought stress

中图分类号:  S722.8

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