Abstract
Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes—ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)—were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.
Highlights
The continual growth of populations and excessive use of chemical fertilizers in agricultural productivities have remarkably and negatively influenced the natural environment: arable lands have decreased, air and water have been polluted, severe desertification has increased, global warming is changes the climate, and frequent natural disasters threaten plant growth [1, 2]
A total of the ten most commonly used reference genes in the reports of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) in graminaceous crops, including ACT, E2, TUBα, TUBβ6, GAPDH, EF-1α, SAMDC, PKABA1, PGK, and HSP90, were chosen as candidates for gene expression stability assessment under various abiotic stresses The specification of primers for all candidate genes was verified via PCR amplification before performing the qRT-PCR reactions
To analyze the expression level of these 10 candidate reference genes under three different experimental conditions, the cycle threshold (Ct) values of all analyzed samples were obtained via qRT-PCR, and the average Ct values of each gene under all experimental groups were calculated
Summary
The continual growth of populations and excessive use of chemical fertilizers in agricultural productivities have remarkably and negatively influenced the natural environment: arable lands have decreased, air and water have been polluted, severe desertification has increased, global warming is changes the climate, and frequent natural disasters threaten plant growth [1, 2]. Among all abiotic stress factors—such as drought, high temperatures, cold, UV radiation, and mechanical injury —salt, cold, and drought stresses are the most regular abiotic stresses, which may be encountered by crops throughout their life spans and which negatively affect growth, yield, and quality of cereal crops [3,4,5]. The focus of research has always been on the abiotic stresses of plants [6]. The genome sequences of many important crops have been recently reported, such as rice and barley [7, 8]. These studies have accelerated the development of genomics and comparative genomics research in crops enormously, and provided a shortcut for revealing the constitution and regulations of plant stress-resistance mechanisms in different species via comparative genomics method
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