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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 16C. 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 3PtrSND1 -- 1R 5 GTCAGAATTC.TTGACAACTG 3

    PtrSND1 2F 5 AGCTGGATCC.TATATCTGTG 3PtrSND1 2R 5 GTCAGAATTC.TTGGCAAGTG 3

    PtrSND1 3F 5 AGCTGGATCC.TGAATCTATC 3PtrSND1 3R 5 GTCAGAATTC.GGCATAATGG 3

    PtrSND1 4F 5 AGCTGGATCC.TGAATCTATC 3PtrSND1 -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/l)

    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 arent 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

    Slide Number 1