Biology Reference
In-Depth Information
AtCycB1;1 cDNA or pDONR221 for H2B and DUO1 and mDUO1 cDNA,
pDONRP4P1R for promoter regions and pDONRP2RP3 for GFP and RFP) via
a BP reaction using BP Clonase II (Invitrogen). The product of BP reactions was
transformed into alpha-select chemically competent cells (Bioline) and all clones
were verified by sequencing.
A multipart LR reaction using LR Clonase plus (Invitrogen) and the des-
tination vector pK7m34GW [32] was used to generate the AtMGH3 marker,
AtMGH3-H2B::GFP. This contains the region upstream of the AtMGH3
coding region [7] driving expression of a H2B::GFP fusion protein, with the
H2B used to give a nuclear GFP signal. The GCS1-GCS1::GFP marker was
constructed by inserting a PCR fragment of GFP into an AflII site in the
16th exon of a Arabidopsis GCS1 genomic DNA fragment in the previously
described binary vector [10]. The AtGEX2-GFP marker, with the GEX2 pro-
moter region driving GFP expression was kindly provided by Shelia McCor-
mick [9].
he vectors DUO1-DUO1::mRFP, DUO1-H2B::mRFP and LAT52-
DUO1::mRFP were also generated using gateway multisite cloning and the vec-
tors pK7m34GW or pB7m34GW [32]. DUO1-DUO1::mRFP uses the DUO1
promoter region to drive expression of a DUO1::mRFP fusion (used to follow
the DUO1 protein during pollen development) while DUO1-H2B::GFP uses
the DUO1 promoter to produce a H2B::mRFP fusion protein (used to follow
the activity of the DUO1 promoter in duo1 pollen). The LAT52 promoter is ac-
tive in the vegetative cell [23] so was used to ectopically express DUO1::mRFP
in the vegetative cell. Vectors to analyse the DUO1 promoter region were also
constructed using gateway multisite cloning.
The DUO1 mRNA contains a functional recognition site for the microRNA
miR159 [21], so for inducible expression of DUO1 a miR159 resistant version of
the DUO1 cDNA was used containing silent mutations in the miR159 binding
site. This was cloned in the vector pMDC7 [33] that contains the XVE estradiol
inducible promoter system [22], using a single part LR reaction and LR Clonase
II (Invitrogen).
For experiments examining the ability of AtCycB1;1 to complement the duo1
division phenotype the vectors pB2GW7 and pH2GW7 [34] were modified to
contain the DUO1 and LAT52 promoters respectively. The 1.2 kb DUO1 and
609 bp LAT52 promoter fragments were amplified from cloned sequences using
restriction tagged oligonucleotide primer pairs. A single part gateway reaction
was then used to clone AtCycB1;1 into the vectors creating DUO1-CycB1;1 and
LAT52-CycB1;1. MGH3-CycB1;1::GFP was generated using a multipart gate-
way reaction.
