Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments

Huan Liu; Yaning Lu; Xiaojing Wang; Xiaowei Wang; Rongchen Li; Cunfu Lu; Xiaozhong Lan; Yuzhen Chen

doi:10.3390/genes13050904

Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments

Genes (Basel). 2022 May 18;13(5):904. doi: 10.3390/genes13050904.

Authors

Huan Liu¹, Yaning Lu¹, Xiaojing Wang¹, Xiaowei Wang², Rongchen Li¹, Cunfu Lu¹, Xiaozhong Lan³, Yuzhen Chen¹

Affiliations

¹ National Engineering Research Center of Tree Breeding and Ecological Restoration, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China.
² Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
³ The Provincial and Ministerial Co-Founded Collaborative Innovation Center for R & D in Tibet Characteristic Agricultural and Animal Husbandry Resources, The Center for Xizang Chinese (Tibetan) Medicine Resource, Joint Laboratory for Tibetan Materia Medica Resources Scientific Protection and Utilization Research of Tibetan Medical Research Center of Tibet, Tibet Agriculture and Animal Husbandry University, Nyingchi 860000, China.

Abstract

Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps.

Keywords: Saussurea laniceps; abiotic stress; hormonal treatment; reference gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Genes, Plant
Hormones
Plants, Medicinal* / genetics
Saussurea* / genetics
Stress, Physiological / genetics
Tibet

Substances

Hormones

Grants and funding

This work was financially supported by The Special Support Plan for the Talent of the Fifth WANREN Plan in China (Nos. 2020-366), the National Natural Science Foundation of China (Grant Nos. U20A20401, 31270737), Tibet Autonomous Region Major Special Science and Technology (Grant No. XZ201901-GA-04), and the grant for Outstanding Team of Graduate Student Tutors from Beijing Forestry University.