Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
INVESTIGATION OF THE TRANSCRIPTIONAL REGULATION OF CANNABINOID SYNTHESIS IN INDUSTRIAL HEMP
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1029698
Grant No.
2023-67013-38996
Cumulative Award Amt.
$600,000.00
Proposal No.
2022-08462
Multistate No.
(N/A)
Project Start Date
May 1, 2023
Project End Date
Apr 30, 2026
Grant Year
2023
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
(N/A)
Non Technical Summary
Industrial cultivation of hemp (Cannabis sativa L.) is currently experiencing a substantive expansion in the US, brought on by federal legal changes and public demand for newly discovered uses of its products, particularly cannabinoids. Cannabinoids have increasingly significant pharmaceutical relevance; they have therapeutic uses as pain medication, alleviating mental disorders, and cancer treatment. However, due to previous restrictions to cannabis research, relatively little is known about the regulation of the biosynthetic pathways for cannabinoid production. Improved understanding of these processes will allow for better selection for, or modification of, plants with particular cannabinoid content. This could increase profit and reduce risk for growers as well as advance further use of cannabinoids in medical treatments.The overall goal of this proposal is to increase our understanding of the regulation of the cannabinoid biosynthetic network in industrial hemp. The objectives to attain this goal are: 1. To map the relationship between factors that turn on or off the genes for the enzymes in the cannabinoid synthesis pathway, 2. To measure the effect of manipulating these factors on the levels of different cannabinoids in cannabis cells, 3. To engineer hemp plants with modified cannabinoid profiles. These objectives will be accomplished by using the latest technologies for genetic modification as well as bioinformatic and metabolic analyses. The results of the proposed project can be translated into development of varieties that help create or meet emerging and future markets and will contribute towards long-term demand for new agriculturally-based industrial products, specifically chemicals of pharmaceutical relevance.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20117301040100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1730 - Hemp;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The long-term goal of this proposal is to increase our understanding of the transcriptional regulation of the cannabinoid biosynthetic network in industrial hemp.This work will make possible the generation of new varieties (either through conventional or new plant breeding technologies) with enhanced or reduced/abolished production of specific cannabinoid compounds. To achieve these goals, our objectives are to study the gene regulatory networks that govern cannabinoid biosynthesis and to measure the effect of manipulation of this regulation on the metabolic profile of cannabis.Objective 1: Construct gene regulatory networks for the 9 candidate TFs using TARGET.Objective 2: Perform nanoparticle-based transient overexpression of these TFs in trichomes.Objective 3: Generate stable overexpression and knock-out lines in tissue culture and plants.
Project Methods
Methods:Transcription factor genes cloned from cDNA or genomic DNA, or synthesized based on available genomic sequenceTransient PEG-mediated transformation of protoplasts for gene regulatory network analysisTransient nanoparticle transformation of floral tissue for gene-expression and metabolite analysisStable Agrobacterium-mediated transformation of callus and whole plantsfor gene-expression and metabolite analysisRNA-seq and qRT-PCR for transcriptional analysisGas- and liquid-chromatography mass spectrometry analysis for metabolite quantificationYeast-one-hybrid analysis for transcription factor-promoter interaction analysis

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:The target audiences reached by our efforts during this reporting period include fellow scientists and students, through efforts including seminars at professional conferences and institutional speaking events, laboratory instruction and formal classroom instruction. Changes/Problems:One of the two graduate students working on this project was awarded a two-semester scholarship, meaningthat we have spent less money so far than originally projected and can either extend the period of work or add to the personnel working on this project. What opportunities for training and professional development has the project provided?Guest lecture about genetic modification to study cannabinoid biosynthsis in industrial hemp given by a graduate student to an audience of undergarduate students in the Virginia Tech School of Plant and Environmental Science Crop PHysiology and Ecology course. Laboratory instruction to teach molecular biology, bioinformatics, plant tissue culture, cell biology, and biochemistry to two graduate students and one undergraduate research assistant. One-week plant transformation and regeneration workshop attendance for one graduate student at theWisconsin Crop Innovation Center. How have the results been disseminated to communities of interest?Poster presentations and seminars given at professional conferences and invited institutional seminar series. Conversations with local (Virginia-based) hemp growers and breeders. What do you plan to do during the next reporting period to accomplish the goals?Clone additional transcription factor and cannabinoid biosynthetic genes. Generation of additional stable transformants (cell suspension and potentially whole plants) with overexpression of candidate transcription factors. Gene regulatory network analysis using transient transformation of protoplasts and RNA-seq analyses. Further metabolite analyses in transgenic cuyltures using LC-MS. Publish two or more papers in peer-reviewed journals.

Impacts
What was accomplished under these goals? Cloning of several transcription factor genes as well as a number of cannabinoid biosynthetic pathway component genes from industrial hemp genotypes. Generation of a cell suspension culture with stable conditional overexpression of a candidate transcription factor as well as a control cell suspension with stable conditional overexpression of green fluorescent protein. RNA-sequencing analysis of the effects of conditional overexpression of a transcription factor, specifically looking at the regulation of cannabinoid biosynthetic genes. Establsihment of Yeast-1-hybrid, qRT-PCR, and transient transformation of protoplasts assays to validate RNA-seq results. Targeted metabolite analyses (LC-MS) of cannabinloid production in transgenic cell suspension cultures, both in cells and spent medium.

Publications