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热带作物学报 ›› 2020, Vol. 41 ›› Issue (5): 955-963.doi: 10.3969/j.issn.1000-2561.2020.05.015

• 生物技术与组织培养 • 上一篇    下一篇

铝胁迫下橡胶苗实时荧光定量PCR内参基因的筛选

马小伟1,2,安锋2,*(),刘子凡1,*(),谢贵水2   

  1. 1. 海南大学热带作物学院,海南海口 570228
    2. 中国热带农业科学院橡胶研究所/农业农村部儋州热带作物科学观测试验站,海南儋州 571737
  • 收稿日期:2019-08-15 修回日期:2019-09-08 出版日期:2020-05-25 发布日期:2020-06-15
  • 通讯作者: 安锋,刘子凡 E-mail:E-mail:an-f@163.com;jiangxilaobiao@163.com
  • 作者简介:马小伟(1994—),男,硕士研究生,研究方向:作物生理与生态。
  • 基金资助:
    海南省自然科学基金高层次人才项目(2019RC326);国家自然科学基金面上项目(31670633);海南省研究生创新科研课题(Hys2019-149)

Screening of Reference Genes for Quantitative Real-time PCR of Rubber Saplings under Aluminum Stress

MA Xiaowei1,2,AN Feng2,*(),LIU Zifan1,*(),XIE Guishui2   

  1. 1. College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China
    2. Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences / Danzhou Investigation & Experiment Station of Tropical Crops, Ministry of Agriculture & Rural Affairs, Danzhou, Hainan 571737, China
  • Received:2019-08-15 Revised:2019-09-08 Online:2020-05-25 Published:2020-06-15
  • Contact: AN Feng,LIU Zifan E-mail:E-mail:an-f@163.com;jiangxilaobiao@163.com

摘要:

铝毒是热带地区酸性土壤上抑制作物生长的主要因素,但目前关于铝活化后对橡胶树生长和产胶的影响及橡胶树耐铝机制的研究很少。为了筛选出橡胶树在铝毒胁迫下稳定表达的内参基因用于实时荧光定量PCR分析,本研究选择了ActinActin718S rRNA40S rRNAYLS8UBC2UBC4GAPDHFPADF等10种常见的基因作为候选内参基因,通过qRT-PCR检测,利用内参基因评价软件geNorm、NormFinder、BestKeeper、Delta Ct算法以及综合评价软件RefFinder对10个候选内参基因的表达稳定性进行评估。综合评价结果表明,铝胁迫后表达稳定性排名前3的内参基因依次分别为UBC440S rRNAFP。进一步选取UBC440S rRNAFP基因和UBC4+40S rRNA+FP基因组合以及稳定性较差的GAPDH基因作为内参基因对橡胶树铝诱导苹果酸分泌转运蛋白基因HbALMT-1HbALMT-2进行相对表达分析,结果显示2个目的基因相对于3个内参基因及其组合均显示出一致的表达水平,而稳定性较差的内参基因GAPDH未能准确地对目的基因的表达量进行校正。综上所述,筛选出UBC440S rRNAFP基因以及UBC4+40S rRNA+FP基因组合可作为铝毒胁迫不同天数下表达最稳定的内参基因,可用于铝毒胁迫下橡胶树相关基因的表达分析。

关键词: 橡胶树, 铝毒, 实时荧光定量PCR, 内参基因

Abstract:

Aluminum toxicity is a major factor inhibiting crop growth on tropical acidic soils. However, the study on the effects of aluminum toxicity on rubber tree growth and latex production is limited, the mechanism of rubber trees tolerate to aluminum has not been reported. To screen the reference genes stably expressed in rubber trees while aluminum stress and elucidate the molecular mechanism of rubber tree aluminum tolerance by real-time quantitative PCR assay, ten candidate internal reference genes, i.e. Actin, Actin7, 18S rRNA, 40S rRNA, YLS8, UBC2, UBC4, GAPDH, FP and ADF were selected in this study. The expression stability was analyzed with geNorm, NormFinder, BestKeeper and Delta Ct and RefFinder. The expression stability of the ten candidate internal reference genes were different. The top three stable reference genes were UBC4, 40S rRNA and FP. Meanwhile, the expression of two aluminum-induced malate transporter genes (HbALMT-1 and HbALMT-2) in rubber trees were verified with UBC4, 40S rRNA, FP, the combination of top three stable genes (UBC4+40S rRNA+FP) and the unstable gene GAPDH. The expression profiles of the two target genes were consistent when normalized by three reference genes and the combinations. The unstable reference gene (GAPDH) failed to standardize the expression data. In conclusion, UBC4, 40S rRNA and FP genes and the combinations were selected as the most stable reference genes, which could be normalized the expression of the relative genes under aluminum stress.

Key words: rubber tree, aluminum toxicity, quantitative real-time PCR, reference gene

中图分类号: 

  • S718.43