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printed by www.postersession.com 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

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Cloning of SND1, Key Regulator of Secondary Cell Wall Biosynthesis Homologous Genes, in Populus trichocarpa

Shari Garrett; Forestry Biotechnology GroupUniversity 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 ExtractionPopulus 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 SequencePtrSND1 – 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 4556 4568 9068 30 min

Temperature (°C) Time 94 5 min

35 cycles 94 45 sec56 45 sec72 90 sec72 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