Arabidopsis thaliana (L.) Heynh.
Creators: name: Guo, Shaobin
name: Wang, Mingdi
name: Xu, Wen
name: Zou, Fuxian
name: Lin, Jingjing
name: Peng, Qin
name: Xu, Wei
name: Xu, Shaohua
name: Shi, Xianai
Publisher: name: Zenodo
Year: 2022
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Magnoliopsida
Brassicales
Brassicaceae
Arabidopsis
Arabidopsis thaliana
A. thaliana

description: 2.1. Expression of UGTs from A. thaliana using the LET-based-TX-TL
system
In order to demonstrate that the LET-based-TX-TL system can be used to screen plant enzymes, we first expressed UGTs from the model plant A. thaliana, whose whole genome sequence is well-characterized. We selected the reviewed group of AtUGTs (See Table 1), which consists of 11 AtUGTs whose catalytic activities have been demonstrated in previous studies (Lim et al., 2004).
As a positive control, we synthesized an AtUGT gene AT5G17050 (AT5G17050-synthesis) from the reviewed group, which encodes for UGT78D2 and has been shown to have the highest glycosyltransferase activity among all the AtUGTs tested (Lim et al., 2004). We extracted RNA from A. thaliana and performed reverse transcription to get cDNA. Then we used this cDNA as a template to amplify the AT5G17050 genes of A. thaliana. After obtaining expressible linear DNAs of AT5G17050-synthesis (obtained by gene synthesis), AT5G17050-cDNA (amplified from cDNA), with either long primers or short primers followed by ligation, we mixed them with the TX-TL extract and buffer to make the reaction mixtures for expression at 29 ◦ C for 8 h and then directly used them to perform glycosylation reactions using quercetin as the substrate. UPLC-MS (Ultra Performance Liquid Chromatography tandem Mass Spectrometry) results showed that the positive control AT5G17050-synthesis and AT5G17050-cDNA exhibited significant glycosyltransferase activity which catalyzed the conversion of quercetin to isoquercitrin (See Supplementary Fig. S1, S 2). This suggested that the TX-TL system can be used to screen active enzymes from plants in vitro. We then test the rest of the reviewed group consisting of 10 AtUGTs. To increase the ligation efficiency of the Golden Gate assembly, we picked the AT2G15490 gene to explore the optimal template concentration in polymerase chain reactions to get gene fragments with the highest quality for the assembly. We found that the cleanest band was obtained when the final template concentration was 10 ng /μL (See Supplementary Fig. S3). So a final template concentration of 10 ng /μL was used to amplify the remaining 10 AtUGTs. We successfully amplified 10 AtUGTs gene fragments using PCR and ligated the gene fragments with a constitutive promoter pOR2-OR1-Pr, a strong ribosome binding site UTR1, a strong terminator T500, and a backbone to generate circular DNAs. We then performed another PCR to get expressible linear DNAs.
In total, we obtained 10 different linear DNAs ready for the TX-TL reaction. Linear DNAs were added to TX-TL extract and buffer to make the reaction mixtures, which were incubated at 29 ◦ C for 8 h. After expressing all 10 AtUGTs in TX-TL, we used the reaction mixtures directly to catalyze the glycosylation of quercetin. UPLC-MS results showed that all of the tested glycosyltransferases exhibited positive glycosylation activity (See Fig. 2, Supplementary Table S1).
description: Published as part of Guo, Shaobin, Wang, Mingdi, Xu, Wen, Zou, Fuxian, Lin, Jingjing, Peng, Qin, Xu, Wei, Xu, Shaohua & Shi, Xianai, 2022, Rapid screening of glycosyltransferases in plants using a linear DNA expression template based cell-free transcription-translation system, pp. 1-9 in Phytochemistry (113007) (113007) 193 on pages 2-3, DOI: 10.1016/j.phytochem.2021.113007, http://zenodo.org/record/8234972