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Cloning of SND1, Key Regulator of Secondary Cell Wall
Biosynthesis Homologous Genes, in Populus trichocarpa
Shari Garrett; Forestry Biotechnology Group
University of Georgia; North Carolina State University
The evolution to produce conducting tissues with rigid secondary cell walls was critical for
plants to be able to dominate the terrestrial habitat of this Earth. These cell walls facilitated the
transport of water and nutrients throughout the plant which in turn allowed them to grow
upright. Research has been done that shows that secondary cell walls can reduce our
dependency on petroleum because it contain a huge bulk of the biomass that can be converted
into fuel. The secondary wall associated NAC domain protein (SND1) is a transcription factor
that is a key regulator in the biosynthesis of the secondary cell wall. SND1 also regulates the
expression of other transcription factors that are also highly expressed. If SND1 is repressed it
causes a reduction of the thickening of the cell wall. If over expressed it results in the
activation of secondary cell wall biosynthesis. Understanding the regulatory mechanisms
controlling cell wall development is an important use in biotechnology.
More research will be done to supplement this project. Homologous genes SND1-1 and SND1-2 will
continued to be worked to achieve a desired outcome as genes SND1-3 and SND1-4. Once all 4
coding regions have been cloned in E. coli protein expression can be quantified. Additional work can
be done to determine more information about SND1. The cloned genes can be used as a template for
RNAi constructs preparation for the knockdown of SND1 from P. trichocarpa . If levels of SND1 are
expressed, researchers can better understand how it affects other genes and what those phenotypic or
genotypic outcomes might be .
Amplification of SND1 Homologues
Fig. 1 Shows agarsoe gel electrophoresis for
amplification of SND1 homologues
I would like to thank Dr. Vincent Chiang, of the Forest Biotechnology Group, for allowing me the
opportunity to work in his lab and providing the necessary materials that went into this project. I
would also like to thank Dr. Quanzi Li for his advice and assistance during the duration of this project.
Zhong, Ruiqin., Richardson, Elizabeth A., Ye, Zheng-Hua. 2007. Two NAC Transcription Factors,
SND1and NST1, function redundantly in regulation of secondary wall synthesis in fibers of
Arabidopsis. Planta 225:1603-1611.
Zhong, Ruiqin., Demura, Taku., Ye, Zheng-Hua. 2006. SND1,a NAC Domain Transcription Factor, Is
a Key Regulator of Secondary Cell Wall Synthesis in Fibers of Arabidopsis. The Plant Cell 18: 3158-
3170.
Plant Cell Walls. Complex Carbohydrate Research Center: University of Georgia. URL
http://www.ccrc.uga.edu/~mao/intro/ouline.htm
cDNA Extraction
Populus trichocarpa xylem DNA was extracted and underwent RT-PCR were cDNA
was established.
Amplification
From Eurofins mwg operon high quality custom oligo set, the following primers were
chosen based on their similar temperature melting, percent guanine and cytosine, and the lack
of problems they might occurring during amplification. A 1X working solution of each primer
and was used during amplification.
Amplifications were performed using the DNA Engine Peltier Thermal Cycler by MJ Research.
PCR Program
The amplifications were analyzed on 1.2% agarose gel electrophoresis. The PCR products were
then purified using the Qiagen PCR Purification Kit.
TA Cloning
The pRSET-A vector and the amplified inserts were prepped for TA cloning and
underwent ligation overnight at 16°C.
Transformation
Top 10 E. coli cells were transformed with the plasmid construct. The cells were then
spread on to LB/Amp/ X-gal plates and were analyzed using the blue-white colony screen.
Individual white colonies were selected and inoculated in LB/Amp broth. The colonies also
underwent colony PCR to screen for correct DNA vector constructs.
Colony PCR Program
The colony PCR products were analyzed on a 1.2% agarose electrophoresis gel.
Mini-Prep
The plasmid DNA was extracted using the Qiagen Mini-prep kit.
Quantification
The DNA concentrations of each gene sample were analyzed using the Thermo
Scientific NanoDrop 2000 Spectrophotometer.
Sequencing
Purified DNA underwent sequencing at the Genomic Sciences Laboratory (NCSU).
INTRODUCTION
MATERIALS AND METHODS
RESULTS CONCLUSION
LITERATURE CITED
ACKNOWLEDGEMENTS
Primer Name Sequence
PtrSND1 – 1F 5’ AGCTGGATCC…..GGTCAATCCC 3’
PtrSND1 -- 1R 5’ GTCAGAATTC….TTGACAACTG 3’
PtrSND1 – 2F 5’ AGCTGGATCC….TATATCTGTG 3’
PtrSND1 – 2R 5’ GTCAGAATTC….TTGGCAAGTG 3’
PtrSND1 – 3F 5’ AGCTGGATCC….TGAATCTATC 3’
PtrSND1 –3R 5’ GTCAGAATTC….GGCATAATGG 3’
PtrSND1 –4F 5’ AGCTGGATCC….TGAATCTATC 3’
PtrSND1 -4R 5’ GTCAGAATTC…..GCATAATGGG3’
Temperature (°C) Time (secs)
94 120
32 cycles 94 45
56 45
68 90
68 30 min
Temperature (°C) Time
94 5 min
35 cycles 94 45 sec
56 45 sec
72 90 sec
72 10 min
Transformation
Colony PCR
Construct DNA Concentration
Colony Number Concentration (ng/µl)
2A 249.4
3A 418.8
3B 469.3
3C 249.0
3D 250.3
4A 328.9
4C 402.5
4D 230.8
Sequencing Results
The colonies that did not contain the desired SND1 sequence are false colonies. Colony PCR reactions
that arise from a transformation can give false positives. There aren’t many reasons why false positives
occur but one reason is that too much unligated insert got transformed into the bacteria. This would
cause a PCR product to be amplified. This could be avoided by using a vector primer and an insert
primer which would prevent the false positives from being amplified. Also, the insert might not be in the
correct orientation within the plasmid. This can be solved by using restriction enzymes to digest the
plasmid and see if the size is correct by means of agarose gel electrophoresis.
Colony Number Sequencing Results
2A Not Sequenced
3A Negative; no insert
3B Positive
3C Negative; no insert
3D Negative; no insert
4A Positive
4C Positive
4D Negative; no insert
FURTHER RESEARCH