Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

Deguchi, Michihito; Potlakayala, Shobha; Spuhler, Zachary; George, Hannah; Sheri, Vijay; Agili, Ruba; Patel, Aayushi; Rudrabhatla, Sairam

doi:10.1371/journal.pone.0260660

Cited by 10 publications

(6 citation statements)

References 51 publications

(58 reference statements)

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“…The gene expression of two cannabinoid pathway genes, CBDAS and THCAS was measured using qRT-PCR (Deguchi et al, 2021 ). Since the CBDAS and THCAS genes have more than 90% homology in their sequences, both sequences were aligned, and a variable region was used to design qRT-PCR primers to avoid off-target effects between them.…”

Section: Methodsmentioning

confidence: 99%

“…The elongation factor 1-α gene was used as a reference gene (Fw: 5'- GCCCTGTCTTTGAGAGCAAC-3', Rv: 5'-CAATCCACTGCTCAATGTGG-3'). Relative gene expression levels of CBDAS and THCAS were calculated using the 2 −ΔΔCt method (Deguchi et al, 2021 ). Statistical analysis was performed using a 1-way ANOVA with Tukey's multiple comparison test (α = 0.05).…”

Section: Methodsmentioning

confidence: 99%

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In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

et al. 2022

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Industrial hemp is a diploid (2n = 20), dioecious plant, and an essential source of various phytochemical productions. More than 540 phytochemicals have been described, some of which proved helpful in the remedial treatment of human diseases. Therefore, further study of hemp phytochemicals in medicine is highly anticipated. Previously, we developed the vacuum agroinfiltration method, which allows the transient gene expression in hemp tissues including female flowers, where cannabinoids are produced and accumulated. In this study, we attempted to alter the composition of total CBD and THC. The RT-PCR and sanger sequence identified eleven copies of the CBDAS gene, two copies of the THCAS gene, and one CBCAS gene. Binary vectors were constructed to overexpress the CBDAS gene and silence the THCAS gene via RNA interference. The Transcript level of the CBDAS gene was increased by more than 10 times than the plants used as a control, which led to a 54% higher total CBD content. The silencing of the THCAS gene led to downregulation of the THCAS gene, with an 80% reduction in transcript levels, and total THC content was reduced to 43% compared with mock plant. These results suggest that hemp vacuum infiltration is highly effective for metabolic engineering of cannabinoids in hemp.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

et al. 2022

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“…Two olivetolic acid cyclase genes ( CsAOC-1 and CsAOC-2 ) and seven prenyltransferase -like genes (LOC115713185, LOC115707809, LOC115713148, LOC115713171, LOC115713205, LOC115713215, and LOC115722991) were identified in the CBDRx reference genome (GenBank GCF_900626175.2), but CsPT4 (LOC115713185) was selected for this study as it was the only one found functional in transgenic yeast ( Luo et al., 2019 ). Amplification of three reference genes, Elongation factor 1 alpha , Serine/threonine-protein phosphatase and Tubulin α -1 ( Guo et al., 2018 ; Deguchi et al., 2021 ), selected based on their stability in C. sativa , was used as an internal control for normalizing the relative expression data. Primer efficiencies were tested by comparison against a standard curve for each gene.…”

Section: Methodsmentioning

confidence: 99%

Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.)

Garrido¹,

Rico

Corral³

et al. 2022

Front. Plant Sci.

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Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids.

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“…XM_030654744.1)-for C. sativa were selected according to Deguchi et al (2021), checked in each cDNA using the quantitative PCR quantifcation (qPCR) and analysed with Visual basic application for Excel (GeNorm) that automatically calculates the gene stability (Vandesompele et al 2002). 18S ribosomal RNA (18S) was then selected as the reference gene for the standardisation.…”

Section: Pip Genes Expressionmentioning

confidence: 99%

Aquaporins-mediated water availability in substrates for cannabis cultivation in relation to CBD yield.

Ortiz-Delvasto¹,

García-Gómez²,

Carvajal³

et al. 2023

Preprint

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Aims The objective of this study was to identify the most suitable substrate for Cannabis sativa L. cultivation based on its effects on water relations and CBD production. Methods Biomass production, physiological parameters, mineral contents, changes in the expression levels of the PIP aquaporins and the concentration of CBD was measured in Cannabis sativa L. (var. Tiborszallasi) plants cultivated on 5 substrates with different physical-chemical characteristic under controlled conditions. Results The substrates available water (AW) was the main factor affecting growth and production. The efficiency of the water use was governed fundamentally by transpiration. S1 and S3 were those in which the plants grew optimally and allows plants to invest energy in secondary metabolites production acquiring high levels of CBD. The plants grown in S2 and S5, composed by coconut fiber and perlite, showed the lowest growth in agreement with low transpiration rates which reduce the water uptake. S5 substrate, with some AW still available, is forcing plants to invest energy in improve water and nutrients transport as observed by the high levels of nutrients in planta and high PIPs expression levels. S4 plants presented the highest inflorescences production and CBD content which can be attributed to plant stress due to the low levels of AW and high pH and electrical conductivity (EC). Conclusion The absorption of water and minerals by plants has been affected by PIP-mediated water transport, playing key roles for an optimal use of the water present in the substrates with specific isoforms involved in this responses.

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Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

Cited by 10 publications

References 51 publications

In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.)

Aquaporins-mediated water availability in substrates for cannabis cultivation in relation to CBD yield.

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