Download Supplemental Data

Supplemental Figure S1. The genomic organization of Dbzds. The Dbzds gene is composed of 12
exons inserted by 11 introns which are franked by canonical donor site GT and acceptor site AG.
The Dbzds promoter possesses several regulatory elements besides traditional promoter conserved
motifs. Their consensus sequences and locations are indicated correspondingly, as well as the
CpNpG Island.
Supplemental Figure S2. Fluorescence images on day 2, 21 and 42 after transformation (400 ×).
Only red fluorescence is observed on day 2 in D. bardawil cells transformed with water (A) or
negative control vector pET after transformation (B). Moderate green fluorescence is observed in
D. bardawil cells transformed with pZET (C) and pZBET (D) as soon as 2 days after
transformation. Stable expression of ble-egfp in pZBET vector transformed into D. bardawil cells
grown in Zecoin selective media is obtained as strong green signal remained to be observed 21
days after transformation (E). Strong green fluorescence sustains in the second generation
transformants with pZBET vector (F). The green and red represent EGFP fluorescence and the
chloroplast autofluorescence, respectively.
Supplemental Figure S3. Positive colonies of transformants, PCR and RT-PCR analysis of
ble-egfp DNA and transcripts in D. bardawil cells electroporated with pZBET. Transformed cells
were spread onto Zeocin selective plate immediately (A). When colonies appeared, single colony
was transferred to selective liquid medium for subculture, and passage cells were spread onto the
selective plate again (B). DNA and RNA were extracted from transformed cells and subjected to
PCR (C) and RT-PCR (D) analysis. PCR and RT-PCR products from day 21 and 42 transformants
are loaded in lanes 2 and 3, respectively. Negative control transformed with pET is loaded in lane
1. Plasmid pZBET is used as template to amplify the target serving as positive control (lane 4).
500 bp DNA Ladder Marker is added to indicate the target fragment (lane M).
Supplemental Figure S4. Cloning strategies for isolating crts genomic DNA including promoter
and terminator regions. For isolation of genomic gene of zds, two consecutive steps of genome
walking PCR were conducted. 1st Genome Walk PCR: according to the 3’ UTR of Dbzds cDNA, a
set of adjacent gene specific primers (g1ZdSP1, g1ZdSP2 and g1ZdSP3) were designed for
isolation of Dbzds full-length gene fragment; 2nd Genome Walking PCR: successively, another
genome walking manipulation was conducted to obtain full-length gene of Dbzds, using another
set of gene specific primers (g2ZdSP1, g2ZdSP2 and g2ZdSP3) designed according to the above
obtained fragment. The overlapped region they shared was highlighted as gray box. Similarly,
gene specific primers (PZdSP1, PZdSP2 and PZdSP3; TZdSP1, TZdSP2 and TZdSP3) for cloning
of promoter and terminator regions of Dbzds were designed to reside in the 5’ and 3’ UTR region
of Dbzds, correspondingly. While cloning the full-length gene and promoter of Dbzds, degenerate
primer AP1 supplied by the Kit was used as a sense primer, g1ZdSP1-3, g2ZdSP1-3 and
PZdSP1-3 as antisense primers correspondingly; conversely, AP1 as an antisense primer and
TZdSP1-3 as sense primers when isolating the Dbzds terminator. Other crts were using similar
tactics. For Dbpds, intermediate genomic DNA was retrieved by semi-nested PCR. The numbers
proximal to the SP primers indicate the extending length of Dbzds gene by LA Taq polymerase
with corresponding SP primers when conducting nested PCRs.