Nicotiana benthamiana Experiment:
The experiment began by extracting total RNAs from the hypocotyl tissue of beet and red-leaf beet with TriPure reagent, which was then used to synthesize complementary DNA (cDNA) through reverse transcription. Then we used RT-PCR for amplifying BvAD6 and BvADH, respectively, from the beet and red-leaf beet cDNA.
Figure.1 Total RNA preparation from cotyledon and hypocotyl of Beet and Redleaf Beet
Figure.2 BvAD6 and CTPBvADH amplification from hypocotyl cDNA of Beet and Redleaf Beet
The amplified BvAD6 and BvADH gene fragments were then cloned into the pKBdCAD6, pKBdTCP19AD6, pKCydCAD6, and pEAQCTPBvADH plasmid vectors to create recombinant plasmids. These recombinant plasmids were introduced into the Agrobacterium tumefaciens strain GV3850 in using electroporation.
Figure.3 Simplified diagrams of vectors
Figure.4 Use Western blot to determine the vector build
Figure.5 The simplified diagram of pEAQBvADH and the determination
Figure.6 The process of optical density test
Figure.7 The process of electroporation
The Agrobacterium strains carrying the desired gene constructs were then used to infiltrate Nicotiana benthamiana (benthamiana) plants through agroinfiltration. Leaf samples were collected from the infiltrated benthamiana plants at 3, 5, and 7 days post-agroinfiltration (dpai). Total protein was extracted from these samples and subjected to protein assays to analyze enzyme activity.
Figure.8 Tobacco after Agro-infiltration
Figure.9 Detect the infection of Agrobacterium
Figure.10 The infected leaves under normal light
Figure.11 Detection of AD6 and ADH expression in N. benthamiana
Figure.12 Detection of AD6 and ADH expression in N. benthamiana
Finally, we used high-performance liquid chromatography (HPLC) method for analyzing and quantifying the L-DOPA from leaves.
Zebrafish Experiment:
- L-DOPA (24~120 hpf) : 0.5 mM / 1.0 mM
- Test: Fertilized zebrafish embryos were cultured in egg water in a petri dish. After 24 hours, the embryos were transferred to a 24-well plate, with 8 embryos per well. The embryos were immersed in 2 mL baths of 0.5 mM and 1.0 mM L-DOPA, with each concentration occupying two wells. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.
- MPTP (48~120 hpf) : 50 μM / 100 μM / 200 μΜ
- Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with 8 embryos per well. Each well was filled with 2 mL of 50 μM, 100 μM, or 200 μM MPTP, with each concentration occupying two wells. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.
- MPTP : 100μM , L-DOPA : 0.5mM
- Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 24 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate. For the Test group, each well received 2 mL of 0.5 mM L-DOPA, with three wells per concentration and 8 embryos per well. For the positive control group, each well was filled with 2 mL of egg water. At 48 hpf, the Test wells were changed to a solution of 2 mL 0.5 mM L-DOPA + 100 μM MPTP, while the positive control wells were changed to 2 mL 100 μM MPTP. Swimming distance was observed at 120 hpf using Danio Vision.
- Due to time constraints and uncontrollable factors such as the occasional low yield or lack of egg production in zebrafish, we have only completed the neuroprotective testing of the chemically synthesized standards. We have not yet been able to conduct comparative testing between the plant-synthesized samples and the chemically synthesized standards.
- L-DOPA (72~120 hpf) : 0.5 mM / 1.0 mM
- Test : Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 72 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, where they were immersed in 2 mL baths of 0.5 mM and 1.0 mM L-DOPA, with each concentration occupying two wells and 8 embryos per well. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.
- MPTP (48~72 hpf) : 200 μM / 400 μM / 600 μΜ
- Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with each well containing 2 mL of 50 μM, 100 μM, or 200 μM MPTP, using two wells per concentration and 8 embryos per well. At 72 hpf, the MPTP bath was replaced with egg water. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.
- MPTP : 400 μM , L-DOPA : 0.5 mM / 1 mM
- Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with each well receiving 2 mL of 400 μM MPTP, using three wells for this concentration and 8 embryos per well. At 72 hpf, the Test group had their bath replaced with L-DOPA, while the positive control group had their bath replaced with egg water. At 120 hpf, swimming distance was observed using Danio Vision, and immunofluorescence staining was performed for further analysis. Additionally, swimming distance and survival rates were recorded at 120 hpf using Danio Vision.
- Due to time constraints and uncontrollable factors such as the occasional low yield or lack of egg production in zebrafish, we have not yet been able to complete the recovery testing of the standards.
The aim of this experiment was to evaluate the therapeutic effect of different sources of L-DOPA (Stednitz et al., 2015) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Kim et al., 2023; Manasa, Chitra, & Tamilanban, 2020; Omar, Kumar, & Teoh, 2023; Stednitz et al., 2015) induced therapeutic effects in a zebrafish model of Parkinson's disease. The following experimental planning was used to investigate the dosage effect, neuroprotective effect (Cronin & Grealy, 2017) and neurorestorative capacity (Cronin & Grealy, 2017) of L-DOPA, as well as the dosage effect of MPTP.
Danio Vision Setup
Zebrafish embryos were placed individually into each well of a 24-well plate, with one embryo per well. The plate was then placed into the Danio Vision system for testing. For the test, the embryos were first given a 5-minute acclimation and rest period. After this, a 3-minute observation period of free swimming behavior was conducted. The Danio Vision camera captured the embryos’ movements during these 3 minutes, recording data such as swimming distance, speed, and acceleration. Swimming distance was selected as the primary parameter for observation and analysis.
Immunohistochemical staining
Parkinson's disease patients experience degeneration of the substantia nigra in the brain, leading to a deficiency of dopaminergic neurons. In zebrafish, the human substantia nigra corresponds to the bilateral ventral brain. Therefore, we aimed to observe the number of dopaminergic neurons in the zebrafish bilateral ventral brain through immunostaining to assess the damage caused by MPTP to these dopaminergic cells.
However, due to insufficient technical expertise and time constraints, we were unable to quantify the results and can only provide photos for reference.
Neuroprotection Experiment
L-DOPA dose test
MPTP Dose Test
Neuroprotection Experiment
Recovery Experiment
L-DOPA dose test
MPTP Dose Test
Recovery Experiment
Assessing Indicators:
1. behavioral analysis (Zhao et al., 2020): we will use Daniovision to assess the swimming behavior of zebrafish, including total path length and swimming speed.
2. Histological analysis (Cronin & Grealy, 2017; Stednitz et al., 2015): Immunohistochemical staining will be performed to assess morphological changes in neurons and expression levels of relevant proteins.
Through this series of experiments, it is expected that the therapeutic effects of L-DOPA can be comprehensively evaluated, including its dose dependence, neuroprotective effects and neurorestorative capacity. Differences in the therapeutic effects of L-DOPA from different sources will also be explored, as well as the possible presence of novel therapeutic compounds in Nicotiana benthamiana extract. This study is expected to provide new insights and potential therapeutic options for the treatment strategy of Parkinson's disease.