Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha

Abstract

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Phototropin perceives temperature based on the lifetime of its photoactivated state

“…The mutated DNA fragments were replaced with the corresponding sequences in pENTR-gMpPHOT using the unique restriction enzyme sites, AgeI/AvrII. The resulting plasmids were mixed with a destination vector, pMpGWB301 (25), and the LR reaction was performed to produce pMpGWB301-MpPHOT DNA fragments for the LOV1 and LOV2 domains of Mpphot were amplified with MpPHOT cDNA as template (26) by PCR with primers (for LOV1: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCAGCAGACCTTTGTGGTC-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATGTGTATTTGCTGACC-3′, and for LOV2: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAGAGTATTGATGA-CAGT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGCTCACTTCC-ATCG-3′) and cloned into the pDONR207 vector by the BP reaction. The C328A mutation for LOV1 and the C628A or V594T mutation for LOV2 were introduced by DpnI-mediated site-directed mutagenesis with the following primers (for C328A: 5′-ATCGGAAGAAACGCTCGATTTCTCCAGGGA-3′ and 5′-TCCCTGGAGAA-ATCGAGCGTTTCTTCCGAT-3′, for C628A: 5′-ATCGGAAGAAATGCTCGGTTTCTGC-AAGGG-3′ and 5′-CCCTTGCAGAAACCGAGCATTTCTTCCGAT-3′, and for V594T: 5′-CGAATTGAGAAGAATTTCACGATTACAGATCCTCG-3′ and 5′-CGAGGATCTGTA-ATCGTGAAATTCTTCTCAATTCG-3′).…”

An adenylyl cyclase with a phosphodiesterase domain in basal plants with a motile sperm system

“…The amplified DNA fragment was cloned into the cloning vector pENTR/D-TOPO (Thermo Fisher Scientific, USA). The resulting plasmid was used for LR recombination by the Gateway technique (Thermo Fisher Scientific, USA) with pMpGWB10456 containing a GUS gene to produce the binary vector pMpGWB-Pcape. The GUS protein should be expressed as a translational fusion with the N-terminal fragment of MpCAPE (Met-1 to His-259).…”

The Liverwort, Marchantia, Drives Alternative Electron Flow Using a Flavodiiron Protein to Protect PSI

“…To generate complementation lines of DMpFlv1, a binary vector, pMpGWB306, harboring a mutated acetolactate synthase gene that confers chlorosulfuron resistance was used (Ishizaki et al, 2015). The coding region of MpFlv1 was amplified from cDNA from Tak-1 through PCR using KOD plus Neo (Toyobo) with the primer set C (Supplemental Table S1) and was then cloned into pENTR/D-TOPO (Thermo Fisher Scientific).…”

“…The resultant MpFlv1 cassette was cloned into pMpGWB306 using LR Clonase II (Thermo Fisher Scientific) according to the manufacturer's protocol. The inserted MpFlv1 was driven by the cauliflower mosaic virus 35S promoter (Ishizaki et al, 2015). Complementation lines were generated by transforming the resulting binary plasmids into regenerating thalli of DMpFlv1, as described previously (Kubota et al, 2013).…”