联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月2日 星期六
农业生物技术学报  2024, Vol. 32 Issue (4): 926-938    DOI: 10.3969/j.issn.1674-7968.2024.04.017
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
杜鹃兰qRT-PCR内参基因的筛选及稳定性评价
张玉琎1, 吉军1, 肖鑫1, 张静怡1, 王利琴1, 高燕燕2, 田宇航1, 张明生1,*
1 贵州大学 生命科学学院/山地植物资源保护与种质创新教育部重点实验室,贵阳550025;
2 贵州医科大学 药学院,贵阳 550025
Screening and Stability Evaluation on qRT-PCR Reference Genes of Cremastra appendiculata
ZHANG Yu-Jin1, JI Jun1, XIAO Xin1, ZHANG Jing-Yi1, WANG Li-Qin1, GAO Yan-Yan2, TIAN Yu-Hang1, ZHANG Ming-Sheng1,*
1 School of Life Sciences/Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China;
2 College of Pharmacy, Guizhou Medical University, Guiyang 550025, China
全文: PDF (2677 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
摘要 杜鹃兰(Cremastra appendiculata)为兰科珍稀药用植物,因其具有较高的药用价值而获得广泛关注。由于存在严重的繁殖障碍,加之人类的掠夺式采挖,导致其资源濒临枯竭,利用现代生物分子技术解决杜鹃兰物种繁殖困难的问题是资源保护的有效途径之一。本研究以杜鹃兰不同器官(根, 茎和叶)和不同萌发阶段的种子(共生萌发和非共生萌发种子)为材料,采用qRT-PCR技术检测肌动蛋白(β-actin, ACT)、翻译延伸因子(translation elongation factor 1 beta, ef-)、亲环蛋白(cyclophilin, CYP)、核糖体蛋白(ribosomal protein, RPL)、核糖体蛋白S8 (ribosomal protein S8, RPS)、泛素C (ubiquitin C, UBC)、微管蛋白(α-tubulin, TUB)共计7个常用管家基因的表达情况,并结合geNorm、NormFinder和BestKeeper软件对候选基因的稳定性进行综合评价,根据分析结果筛选适于杜鹃兰qRT-PCR的内参基因;并通过目的基因赤霉素氧化酶基因(GA 3-beta dioxygenase, GA3ox)和凝集素蛋白编码基因10 (lectin protein coding gene 10, Lectin10)对内参基因的稳定性进行验证。研究发现,在杜鹃兰不同处理条件和不同组织下均可引入双内参基因进行实验;在不同器官中,RPLef-的表达相对一致,稳定性较好,适合作为内参基因;在不同萌发阶段的共生萌发种子中,UBCTUB表达最为稳定,而不同萌发阶段的非共生萌发种子中最稳定的内参基因为RPLef-。分别选择相对稳定的UBCTUB作为内参基因时,目的基因GA3oxLectin10在不同萌发阶段的共生杜鹃兰种子中相对表达变化趋势基本一致,而稳定性较差的ACT并没有对目的基因的表达分析进行有效校正,其数据出现明显偏差。本研究为不同研究条件下杜鹃兰相关基因的表达分析提供了校正和标准化基因,有效提高了后续研究的准确性和可靠性。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
张玉琎
吉军
肖鑫
张静怡
王利琴
高燕燕
田宇航
张明生
关键词 杜鹃兰共生萌发非共生萌发qRT-PCR内参基因    
Abstract:As a rare medicinal plant of orchid family, Cremastra appendiculata has been widely concerned for its high medicinal value, but its resources are depleted by severe reproductive barriers and predatory human extraction, using modern biomolecular technology to solve the reproduction difficulties of C. appendiculata species is one of the effective ways of resource conservation. In this study, different organs (root, stem and leaf) and seeds at different stages (the symbiotic and non-symbiotic germination) of C. appendiculata were used as research materials, qRT-PCR was used to detect the expression of 7 commonly used household genes of β-actin (ACT), translation elongation factor 1 beta (ef-), cyclophilin (CYP), ribosomal protein (RPL), ribosomal protein S8 (RPS), ubiquitin C (UBC), α-tubulin (TUB). The stability of candidate genes was comprehensively evaluated by geNorm, NormFinder and BestKeeper software, and finally the reference genes suitable for qRT-PCR of C. appendiculata were selected. The stability of the reference genes was verified by the target genes GA 3-beta dioxygenase (GA3ox) and lectin protein coding gene (Lectin10). The result showed that 2 reference genes could be introduced into different organs and treatments of C. appendiculata; The expression of RPL and ef- in different organs was relatively consistent, with good stability, and suitable as reference genes; The expression of UBC and TUB were the most stable in the symbiotic germinated seeds at different germination stages; RPL and ef- were the most stable reference genes in the non-symbiotic germination seeds at different germination stages. When the relatively stable UBC and TUB genes were used as internal reference genes, the target genes GA3ox and Lectin10 showed similar expression trends in the seed of C. appendiculata at different symbiotic germination stages, while the poorly stable ACT genes could not effectively correct the expression analysis of target genes, and the data showed extremely obvious deviations. This study provides correction and standardization genes for the expression analysis of related genes in C. appendiculata under different research conditions, which would effectively improve the accuracy and reliability of subsequent studies.
Key wordsCremastra appendiculata    Symbiotic germination    Non-symbiotic germination    qRT-PCR    Reference gene
收稿日期: 2023-03-27     
ZTFLH:  S3  
  Q94.336  
基金资助:国家自然科学基金面上项目(32270311); 贵州省山地农业关键核心技术攻关项目(GZNYGJHX-2023011); 贵州省科技计划重大专项课题(黔科合平台人才[2017]5411-06); 国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13); 贵州省中药材现代产业技术体系建设项目(GZCYTX-02)
通讯作者: *mshzhang@163.com   
引用本文:   
张玉琎, 吉军, 肖鑫, 张静怡, 王利琴, 高燕燕, 田宇航, 张明生. 杜鹃兰qRT-PCR内参基因的筛选及稳定性评价[J]. 农业生物技术学报, 2024, 32(4): 926-938.
ZHANG Yu-Jin, JI Jun, XIAO Xin, ZHANG Jing-Yi, WANG Li-Qin, GAO Yan-Yan, TIAN Yu-Hang, ZHANG Ming-Sheng. Screening and Stability Evaluation on qRT-PCR Reference Genes of Cremastra appendiculata. 农业生物技术学报, 2024, 32(4): 926-938.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.04.017     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I4/926
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn