Solanum lycopersicum Cryptochrome 1 Expression in

Response to Short and Long Photoperiods

Patrick Stumps
March 15, 2019
 Stumps 2

Introduction

Within the vast quantity of genes discovered by humanity, there lies a small group at the helm
for the perception of the daily rhythm of night and day. While their current function in humans
have avoided elucidation, previous studies have shown their presence in humans may have once
been responsible for magnetosensitivity (Foley et al. 2011). Their role in the lives of other
organisms is much more prominent, however, as they have been shown to not only help control
plant growth (Pedmale et al. 2015) but also contribute to successful migration patterns of the
monarch butterfly (Danaus plexippus) as they mediate their circadian clock (Zhu et al. 2008).
Their long existence throughout the timeline of life perhaps provides some evidence into the
importance these genes play for not only man, but also animals and plants. These genes, called
cryptochromes, are blue light photoreceptors and are thought to have evolved from their DNA
repairing ancestors’ photolyases. Cryptochromes 1 (cry1) and 2 have previously shown to
contribute to nitrogen regulation, as well as flowering of the Arabidopsis plant (Perrotta et al.,
2000; El-Din El-Assal et al., 2003) while also playing a critical role in tomato plant development
(Ninu et al. 2005). Cryptochromes also play an important role in gene expression in
Chlamydomonas reinhardtii, as previous research has shown (Beel et al. 2012).

Light itself provides extremely important environmental information for a large variety of
organisms on earth, especially plants, as it acts as their main energy source. Different light
periods can have astronomical effects on plants, showing differences flowering, budding, and
tuberization (Jackson 2009). Light has also been shown to regulate alternative splicing various
Arabidopsis genes, while even affecting alternative splicing in plant roots (Petrillo et al. 2014).
While previous research has commonly determined how cryptochromes affect expression of
other genes, (Lopez et al. 2012, Beel et al. 2012), only a few have been conducted to determine
how photoperiods affect cry1. This study determined relative cry1 expression ratios using
qualitative PCR followed by next-generation sequencing methods to confirm gene amplification.
It was determined that cry1 has a 4.89-fold increase in expression in S. lycopersicum exposed to
long photoperiods versus short photoperiods.
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Methods

Photoperiod Exposure/RNA Isolation

Short photoperiod tomato plants were exposed to a photoperiod of 12 hours of light and 12 hours
of dark, and long photoperiod tomato plants were exposed to 16 hours of light and 8 hours of
dark prior to RNA isolation. RNA was obtained from .098g of both long and short photoperiod
exposed plant. RNA was then purified using the Qiagen RNeasy kit with the protocol provided
by the manufacturer, along with a DNAse I treatment. I deviated from the protocol by spinning
produced lysate in the QIAShredder at 14,000 RPM instead of 10,000 RPM. RNA concentration
was quantified using UV Spectrophotometry with the Thermo Scientific NANODROP 2000c. A
total of 500ng of isolated RNA was then subjected to a 1.2% formaldehyde agarose gel for
visualization with a NEB ssRNA ladder. I measured less than 80ngL-1 of isolated RNA for the
RNA isolated from the long plant, so I proceeded with the rest of the experiment using RNA
samples from a different lab group.

cDNA synthesis/qPCR
Reverse transcription reactions were performed using the Quanta Biosciences qScript cDNA
synthesis kit in accordance with the provided protocol. A cDNA synthesis mix was created with
500ng of RNA. Quantitative PCR was then performed according to the iQ SYBR Supermix
manual and fluorescence was generated with SYBER Green I dye. Each set of reactions
consisted of 3 short/long cDNA reactions and 3 controls, with each control being one consisting
of nuclease free water, one with RNA only, and one with RNA without the DNAse treatment.
Each PCR reaction, a 1/5th dilution of synthesized long and short cDNA. cry1 forward primer
used was 5’-TCCACTTCTTCCTCCCAAA-3’, reverse primer used was 5’-
CATTGCTTCCTTTCTCTGATTC-3’ both with a Tm of 58C. The amplicon size for the cry1
primers was 100bp. ef1 forward primer used was 5’-ATTGGAAACGGATATGCTCCA-3’,
reverse primer was 5’-TCCTTACCTGAACGCCTGTCA-3’, both with a Tm of 60C. The
amplicon size for ef1 primers was 101bp. Comparison of long to short RNA expression was
calculated using the 2Ct or Livak method. After qPCR each reaction was visualized with a 1.5%
agarose gel with a NEB 100 bp DNA ladder.
 Stumps 4

Large-scale PCR
Large-scale PCR was performed on synthesized cDNA for both cry1 and ef1. The primers used
for the PCR experiment were gene specific and were composed of the following sequences: cry1
forward: 5’-TCCACTTCTTCCTCCCAAA-3’, cry1 reverse: 5’-
CATTGCTTCCTTTCTCTGATTC-3’, ef1 forward: 5’-ATTGGAAACGGATATGCTCCA-3’,
ef1 reverse: 5’-TCCTTACCTGAACGCCTGTCA-3’. The Tm value for both cry1 primers was
58C while the Tm value for the ef1 primers was 60C with an amplicon size of 100bp for cry1
and 101bp for ef1. The PCR reaction was then visualized with a 2% agarose gel.

Cloning
cry1 and ef1 were cloned using DH5 (Escherichia coli). 6.5ng of large-scale PCR was ligated
a 50ng pDrive Cloning vector and inserted in-between the EcoRI restriction enzyme sites within
the LacZ gene on the located on the vector. The two different vectors containing cry1 and ef1
were then transformed into DH5. The first round of transformations were unsuccessful, so a
second set of transformations retrieved from another lab produced with the same parameters
were screened.

Colony PCR
Colony PCR was performed using 5 white colonies and 1 blue for both cry1 and ef1
transformed DH5 cells. The PCR reactions were run with a 1:2 concentration of transformed
DH5 template DNA using M13 primers specific for the lacZ gene. The M13 forward primer
sequence used was 5’-TGTAAAACGACGGCCAGT-3’ and the M13 reverse primer was 5’-
CAGGAAACAGCTATGAC-3’. The gel was then visualized with a 1.5% agarose gel with a
NEB 100bp ladder. The same transformed colonies used in the colony PCR reaction were
streaked on LB + Amp + IPTG + X-Gal plates and cultured overnight. Two white colonies and
one blue colony were then inoculated.
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Plasmid purification / Restriction Digest

Plasmid DNA from inoculated colonies chosen in the colony PCR experiment was purified
according to the Promega wizard Miniprep Manual, with the exception of using 30L of
nuclease free water instead of 50L for the elution step. A restriction digest was then performed
with the restriction enzyme EcoRI using a total of 500ng of plasmid DNA. The restriction digest
reactions were then visualized using a 2% agarose gel.

Sequencing
500ng of purified plasmid DNA with M13 forward and reverse primers were sent to Berkley for
sequencing. The returned sequencing data were trimmed to remove unknown Ns from the ends
of the sequence using the software 4Peaks. The software Geneious (Kearse et al., 2012) was then
used to generate a reverse complement from the returned cry1 and ef1 sequence created from
the reverse M13 primer as the sequences produced from the forward M13 primers were not
successfully sequenced. The sequences were aligned, a consensus sequence was generated and
was then used to search the NCBI nucleotide database within the S. lycopersicum genome.
 Stumps 6

Results

RNA isolation from Solanum lycopersicum

To determine cry1 ratio in S. lycopersicum I quantified isolated RNA from a leaf of both long
and short photoperiod exposed plants. For the long photoperiod exposed control, the total RNA
concentration was 62.3ngL-1 while the A260/A280 absorbance ratio was 2.15. For the short
photoperiod exposed plant, the total RNA concentration was 131.1ngL-1 with a measured
A260/A280 ratio of 2.14. The samples were then run through a gel for visualization. Figure 1
shows lane 8 containing the long photoperiod exposed sample shows signal while lane 9
containing the short photoperiod does not show signal.

Synthesis of S. lycopersicum cDNA and qPCR of cry1 and ef1

Our previous data in figure 1 showed RNA was isolated from the sample, therefore cDNA
synthesis performed to generate a cDNA library for qPCR. Random hexamers and oligo dTs
were used to prime the reverse transcriptase. For qPCR, the primers used were designed to
amplify a 100bp fragment for cry1, while ef1 primers were designed to generate a 101 bp
fragment size. Figure 2 shows the resulting gel analysis from qPCR. Row 1 represents amplified
product produced with ef1 primers and row 2 represents amplified product produced with cry1
primers. Lanes 1-3 contained samples derived from the short photoperiod and lanes 4-6
contained samples from the long photoperiod and signal is present in all lanes near 100bp.
Signal is present in the negative control, lane 8, and RNA control, lane 9, for ef1. Smearing is
present in lane 9, and dark banding is present in lane 10 for cry1 and ef1. cry1 lane 7 contained
light banding relative to the rest of the signal and lane 8 contained smearing. Band was present in
lane 20 with no other signal present.

Table 1 shows the Cq and melt temp data for their respective lanes in figure 2. Amplification
curves for cry1 (figure 3A) and ef1 (figure 3C) show little variance, except for two outliers
shifted to the right in figure 3C. Melt peak data for cry1 (figure 3B) and ef1 (figure 3D) also
show little variance, except for two outliers that only just pass 600 -d(RFU)/dT threshold in
figure 3D.
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Large Scale PCR of cry1 and ef1

In order to confirm whether the genes amplified during qPCR were correct, large-scale PCR was
performed using gene specific primers. These primers were designed to amplify a 100bp
fragment for cry1 and a 101bp fragment for ef1. Figure 4 contains the results from the
performed large-scale PCR. Lane 2 contained the PCR reaction performed with primers specific
for cry1 and lane 4 contained the PCR reaction performed with primers specific for ef1. Lanes
3 and 5 controlled for template DNA presence and contained water and primers for their
respective fragments (lane 3 for cry1 and lane 5 for ef1) with no template cDNA. All lanes
contain signal at around 100bp.

Colony PCR
cry1 and ef1 were ligated into the Eco RI cut site within the LacZ gene of the p-Drive
transformation vector and ligated vectors were transformed into DH5. Because these
transformations produced both white and blue colonies with ambiguous color presence, colony
PCR was performed with LacZ specific primers to determine insert presence. Figure 5 shows the
results of the colony PCR experiment. The expected band size was approximately 343 for cry1
insert, 344 for ef1 insert, and 244 for no insert. Lane 1 contained the NEB 100bp molecular
weight standard. Lanes 2-6 contained PCR product from cry1 fragment ligated transformations.
Lanes 8-11 contained ef1 fragment ligated transformations. To control for cry1 fragment
presence, lane 7 contained PCR product from a blue colony which did not contain ligated
fragment. Lane 13 served a similar purpose contained PCR product from a blue colony and
controlled for ef1 fragment presence. Lane 12 contained PCR product from colonies which
transformed an unsuccessful fragment ligation reaction.

Restriction Digest
Once it was apparent which colonies successfully ligated fragments and which did not, a
restriction digest was performed on plasmid DNA from cultures inoculated from the colony PCR
reaction to determine if insert was present. Figure 6 shows the gel analysis of the restriction
digest. Lane 1 contained the molecular weight standard NEB 100bp DNA ladder. Successful
 Stumps 8

restriction digest fragments had an expected size of 112bp for cry1 and 113bp for ef1. Lanes 2
and 3 contained the restriction digest performed on cry1 transformed inoculated colonies while
lanes 5 and 6 contained ef1 transformed inoculated colonies. Lanes 4 and 7 controlled for insert
presence and contained the restriction digest performed with inoculated colonies that did not
contain insert their respective insert (lane 4 for cry1 and lane 7 for ef1).

Sequencing
Because the restriction digest confirmed the inoculates that contained insert, their plasmid DNA
was purified and sequenced. Purified plasmid DNA was then sent to Berkeley for sequencing
with M13 primers. Both cry1 and ef1 returned sequences generated from a forward and reverse
primer. A reverse complement was created from the sequence generated by the reverse primer
for both cry1 and ef1, the sequences were aligned with their respective forward primer
sequence, and the generated consensus sequence was used to search the NCBI nucleotide
database against the S. Lycopersicum genome. The cry1 consensus sequence showed a bit score
of 178, 97/98(99%) identity, an e-value of 3e-43, and 0/98 gaps (0%). The ef1 consensus
sequence showed a bit score of 182, 97/98 (98%) identity, an e-value of 2e-44, and 0/102 gaps
(0%).
 Stumps 9

Discussion

The goal of these experiments was two-fold. First, to determine cry1 expression ratios in short
vs. long photoperiod grown tomato plants, and second, to confirm the genes analyzed from the
determined expression ratio were cry1 and ef1. The approach to this started with extraction of
RNA from tomato plant leaves. Once RNA was isolated from lysed S. lycopersicum cells, it was
important to determine the quality of the RNA in order to move forward to the next step. Lane 8
of figure 1 shows the visualization of the extracted RNA. RNA in lane 8 is likely of succinct
quality due to the presence of 25S and 18S ribosomal RNA bands. The smearing below the 18S
band could be attributed to slight RNA degradation (Wieczorek et al. 2012) which would likely
explain the low concentration (62.3 ngL-1) determined by the nanodrop analysis of the RNA.
Although lane 9 in figure 1 contains nothing, nanodrop analysis showed a high RNA
concentration of 131.1 ngL-1 with an A260/280 ratio of 2.14 suggesting what was extracted was
not quite pure RNA, but more likely RNA than not. This could suggest the lack of signal was due
to pipetting error rather than RNA degradation.

The previous data showed RNA was present and of sufficient quality, therefore generation of a
cDNA library was required for the next step in determining cry1 expression ratios. The cDNA
generated via reverse transcription was subjected to qPCR, shown in figure 2. The presence of
signal near the 100bp mark in lanes 1-6 for both cry1 and ef1 indicate that the sequence
amplified was likely correct and qPCR was successful, as expected amplicon size was 100bp for
cry1 and 101bp for ef1. Lanes 7 and 8 for ef1 showed some signal near 100bp indicating that
some contamination was present. However, the Cq values for these lanes shown in Table 1 are
extremely high (39.02 and 37.95 for ef1 lanes 7 and 8 respectively) compared to the rest of the
Cq values in ef1, and therefore can be considered trivial (Bustin et al. 2004). Additionally, lane
8 which controlled RNA presence contained smearing for both ef1 and cry1, indicating RNA
was present for cDNA synthesis. Lane 9 lacks signal above 100bp for ef1, therefore no or very
little genomic DNA was present that could have affected other lanes. Lane 9 for cry1 contains
some signal above 100bp around 600bp which could indicate some genomic DNA was present,
however, the melt peaks for cry1 in figure 3 do not show any peaks shifted to the left which
 Stumps 10

passed threshold, indicating that not enough was present to affect the Cq values of the other
reactions (Taylor et al. 2010). Indeed, the melt peaks for both cry1 and ef1 show little variance
indicating that the same amplicon was likely produced for each reaction (Taylor et al. 2010).

The relative amounts of RNA added for each qPCR reaction can vary greatly, as it can not only
be difficult to get an exact amount of RNA, but small differences in amounts of RNA added prior
to qPCR can cause great variance. Other factors like reverse transcription efficiency and RNA
quality can also affect the outcome of qPCR and make gene expression ratios difficult to
determine. Because of this, it is important to normalize gene expression against a reporter or
housekeeping gene. Previous research has shown ef1 to have stable expression in many
different organisms under stress, including S. lycopersicum, and was therefore chosen to as the
reporter for the qPCR experiment (Lacerda et al. 2015, Tang et al. 2017, Ma et al. 2013).
Relative gene expression ratio of cry1 compared to the reporter ef1 was calculated using the
Livak method (Livak et al. 2001).

1. ∆𝐶𝑞(𝑡𝑒𝑠𝑡) =C𝑞(target, test) - Cq(reference, test)

2. ∆𝐶𝑞(calibrator) =C𝑞(target, calibrator) - C𝑞(reference, calibrator)
3. ∆∆𝐶𝑞 = ∆Cq(test) - ∆Cq(calibrator)
4. 2-Cq = normalized expression ratio

Long Cq means were used as test variables while short Cq means were used as calibrator
variables, with cry1 as the target and ef1 as the reference. The first and second equations allow
for normalization of the target gene to the reference gene, while the calculation of Cq allows
for normalization of Cq of the test sample to Cq of the calibrator sample. The use of this
method renders the relative amount of RNA added for each reverse transcription reaction within
qPCR insignificant so that the fold-change expression ratio is not affected (Livak et al. 2001).
Future research could plot Cq(test) versus various dilutions of cDNA qPCR product. If the
absolute value of the slope of the regression line is near zero, the Cq value would have been
even further validated (Livak et al. 2001). The results of the Livak calculation showed a 4.89-
fold increase of cry1 in long versus short photoperiod grown S. lycopersicum.
 Stumps 11

Although the qPCR analysis gel showed signal in agreement with the expected band length, it
was not for certain what exactly was amplified, and further confirmation of the sequence
amplified by the gene specific primers was required. This was in a multitude of steps. The first
step in this process was to generate a large amount of the fragments amplified in qPCR for
insertion into a vector. There are many different ways to generate large amounts of a specific
product, however, previous research has shown large-scale PCR as an effective low-cost solution
to product generation (Wang et al. 2017). The results of the large-scale PCR experiment (Figure
4) showed signal around 100bp for lanes 2 and 4, indicating that the fragments amplified were
present. Although lanes 3 and 5 controlled for cDNA contamination, it was clear that some
fragment of the same size were amplified for both.

The products from the large-scale PCR reaction were then ligated into the pDrive Cloning Vector
within the EcoRI cut site, which simultaneously disrupted the LacZ gene on the vector. Ligation
of the fragments into this vector was important for its unique restriction enzyme site, the
presence of Kanamycin and Ampicillin for transformant selection, and disruption of the LacZ
gene for colony screening. The ligated vectors were transformed into DH5 competent cells, and
a second set of transformations (see methods) were screened for white and blue colonies.
Because the fragment was purposely inserted to disrupt the LacZ gene on the vector and interfere
with DH5’s ability to produce -galactosidase and metabolize X-gal, colonies which were blue
were known to not contain the insert while colonies which were white did (Juers et al. 2012).
This was further confirmed by the colony PCR results (figure 5), in which the white colonies that
did contain insert in lanes 2-6 and lanes 8-11 show signal just above 300bp. Expected amplicon
size was 343bp for cry1 and 344bp for ef1.

The colony PCR reaction showed the ligations were successful, therefore colonies which were
known to contain insert as determined by the colony PCR were plated a second time. However,
the color of these transformations was ambiguous due to some amount of blue color being
present for nearly all of the colonies present. The colony PCR eliminated this ambiguity as its
results suggested that insert was being expressed, even though the color of the colony was blue.
The presence of blue colonies containing the insert could be due to insufficient amounts of insert
 Stumps 12

used during ligation, or self-ligating vectors without insert transforming into DH5.
Additionally, medium degradation could have also contributed to blue color produced by DH5
(Banerjee et al. 2010).

The restriction digest of the inoculated colonies containing insert further eliminated the color
ambiguity of the transformed colonies plated as their purified plasmid DNA showed signal
around 100bp for both cry1 and ef1 inoculated colonies in lanes 2, 3, 5, and 6 (figure 6). The
expected band sizes for cry1 was approximately 112bp and 113bp for ef1. Additionally, lanes 4
and 7 controlling for insert presence confirmed that the plasmid DNA from the suspected blue
colonies from the original transformation did not in fact contain insert.

Once the restriction digest data had suggested insert presence in the plasmid DNA, the samples
were sequenced with M13 primers specific for the LacZ gene. Once a consensus sequence was
generated from the sequence generated from the reverse primer, the reverse complement of that
sequence, and the sequence generated from the forward primer, the sequence was searched on
the NCBI nucleotide database against the S. lycopersicum genome. The generated consensus
sequence for cry1 indicated a 1 in 3.0*1043 chance of the alignment occurring by chance, and the
sequence for ef1 indicated a 1 in 2.0*1043 chance of the alignment occurring by chance
(Madden et al. 2013). These data suggest that the sequences generated by qPCR were indeed
cry1 and ef1, allowing further reinforcement the of the hypothesis that photoperiod length
affects cry1 expression in S. lycopersicum.

Previous studies have shown increased hepatic cry1 gene expression in long photoperiod (12
hour) versus short photoperiod (6 hour) exposed mice (Mariné-Casadó et al. 2018). However,
separate studies have also shown Arabidopsis cry1 mRNA expression to be unaffected by light
conditions (Lin et al. 1998; Ahmad et al. 2002). Other studies done have shown expression of
cry1 mRNA in tomato plants to remain constant regardless of light period (Lin et al. 2002). It
shows that more research is necessary to determine cry1 expression ratios in long versus short
photoperiod plants.
 Stumps 13

This study only determined expression between one long photoperiod exposed plant and one
short photoperiod exposed plant. Future research could incorporate several plants or several
different species of plants to evaluate cry1 expression ratios. Additionally, the use of several
reporter or housekeeping genes could also benefit this research by allowing further normalization
of cry1 expression ratios. Expression ratios of cry1 could have been analyzed for more than two
different photoperiod length exposed plants. Gene expression ratios could have also been
determined in different methods as well.
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Figures/Tables

Figure 1. Formaldehyde Agarose gel analysis of RNA isolated from Solanum Lycopersicum.
Lane 1 contains molecular weight standard (NEB 10kBP DNA ladder). Lanes 2-7 contain RNA
isolated from different experiments. Lane 8 contained RNA isolated from the long photoperiod
plant. Lane 9 contained RNA isolated from the short photoperiod plant.

Figure 2. Agarose gel analysis of qPCR products with cry1 and ef1 specific primers.
DNA was separated using electrophoresis on a 1.5% agarose gel. Top row contains qPCR
product produced from ef1 primers, bottom row contains qPCR product produced with cry1
primers. Lane L contains molecular weight standard (NEB 100bp DNA ladder). Lanes 1-3
contain qPCR product from short photoperiod S. lycopersicum and lanes 4-6 contain qPCR
product from long photoperiod S. lycopersicum. Lane 7 controlled for cDNA contamination.
Lane 8 contained RNA and controlled for successful reverse transcription reactions. Lane 9
contained RNA not treated with DNAse and controlled for genomic DNA presence.
 Stumps 15

Table 1. qPCR Cq and Melt temp data produced from SYBR green fluorescence emissions.
Numbers next to the dash represent the in the target column represent the lane number in figure
2. Sample column represents the sample present in that lane. Melt temp corresponds to the melt
temp peak for the qPCR product. Cq values correspond to the Cq value determined for that qPCR
product. Cq mean represents the mean Cq value calculated for the 3 different long and short
samples.
Target Sample Melt Temp Cq Cq Mean
cry1-1 Short 78.50 32.32
cry1-2 Short 78.50 31.11 31.22
cry1-3 Short 78.00 30.23
cry1-4 Long 78.00 29.18
cry1-5 Long 78.50 29.22 28.93
cry1-6 Long 78.50 28.38
cry1-7 No DNA N/A 0
cry1-8 RNA N/A 0
cry1-9 No DNase 78.00 30.66
ef1𝛼-1 Short 83.00 31.25
ef1𝛼-2 Short 83.00 29.62 29.89
ef1𝛼-3 Short 83.00 28.79
ef1𝛼-4 Long 83.00 29.62
ef1𝛼-5 Long 83.00 28.54 28.51
ef1𝛼-6 Long 83.00 27.37
ef1𝛼-7 No DNA 82.50 38.02
ef1𝛼-8 RNA 82.50 37.95
ef1𝛼-9 No DNase 83.00 27.01
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A C

B D
Figure 3. qPCR Amplification curve and Melt Peak data for cry1 (3A, 3B) and ef1 (3C,
3D).
Amplification curve (3A and 3C) horizontal axis represents the cycle number and vertical axis
represents the relative fluorescence units (RFU). Melt peak (3B and 3D) horizontal axis
represents temperature and vertical axis represents the negative derivative of relative
fluorescence with respect to temperature.
 Stumps 17

Figure 4. Agarose gel analysis Large-scale PCR products with gene specific primers.
PCR products were separated using electrophoresis on a 2% agarose gel. Lane 1 contained
molecular weight standard NEB 100bp DNA Ladder. Lane 2 contained PCR product using cry1
gene specific primers. Lane 4 contained PCR product created using ef1 specific primers. Lanes
3 and 5 controlled for unsuccessful large-scale PCR reactions.

Figure 5. Colony PCR products generated with M13 LacZ specific primers.
PCR products were separated using electrophoresis on a 1.5% agarose gel. Lane 1 contained
molecular weight standard (NEB 100bp DNA ladder). Lanes 2-6 contained PCR products from
cry1 ligated p-Drive transformed white colonies. Lanes 8-11 contained PCR products from ef1
ligated p-Drive transformed white colonies. Lane 12 contained PCR product from a colony
which did not successfully transform fragment ligated p-Drive. Lanes 7 and 13 controlled for
unsuccessful fragment ligation and transformation.
 Stumps 18

Figure 6. EcoRI restriction digest of plasmid DNA purified from inoculated DH5
cultures.
DNA was separated using electrophoresis on a 2% agarose gel. Plasmids were cut with
restriction enzyme EcoRI. Lane 1 contained molecular weight standard (NEB 100bp DNA
ladder). Lanes 2 and 3 contained fragment removed from cry1 ligated p-Drive transformed
colonies. Lanes 5 and 6 contained fragments removed from ef1 ligated p-Drive transformed
colonies. Lane 4 controlled for unsuccessful cry1 ligation. Lane 7 controlled for unsuccessful
ef1 ligation.

B
Figure 7. Consensus sequence NCBI nucleotide database search results against Solanum
lycopersicum.
(A) Consensus sequence from cry1 showed a bit score of 178, an expect value of 3*10-43, 97/98
identities (99%), and 0/98 gaps (0%).
(B) Consensus sequence from ef1 which showed a bit score of 182, an expect value of 2*10 -44,
100/102 identities (98%), and 0/102 gaps (0%)
 Stumps 19

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