By convention, single strands of DNA and RNA sequences are written in a 5′-to-3′ direction except as needed to illustrate the pattern of base pairing.

DNA Directionality

Terminology

Kanamycin

Kanamycin is used in molecular biology as a selective agent most commonly to isolate bacteria (e.g., E. coli) which have taken up genes (e.g., of plasmids) coupled to a gene coding for kanamycin resistance (primarily Neomycin phosphotransferase II [NPT II/Neo]).

Bacteria that have been transformed with a plasmid containing the kanamycin resistance gene are plated on kanamycin (50-100 μg/mL) containing agar plates or are grown in media containing kanamycin (50-100 μg/mL).

Only the bacteria that have successfully taken up the kanamycin resistance gene become resistant and will grow under these conditions.

As a powder, kanamycin is white to off-white and is soluble in water (50 mg/mL).

8-oxo-dG

A frequently used biomarker of oxidative DNA damage and can be detected by immunological techniques using the specific anti-8-oxo-dG antibody (Yoshida et al., 2002).

In the oxidized DNA induced by ROS, the 8-oxo-G combines with a deoxyribose in the deoxyguanosine, gives rise to 8-oxo-dG.

Methodology

Plant Materials and Growth Conditions

Growth

A. thaliana (ecotype Columbia-0) plants were grown routinely in a greenhouse under 22±1 °C with a light/dark regime of 16 h light/8 h dark.

Sterilized seeds were sown on Petri dishes containing half-strength Murashige and Skoog (MS) medium (Duchefa, Haarlem, The Netherlands) and then stratified at 4 C for 2 days.

After stratification, seeds were germinated under the same growth conditions as above and seedlings were transferred to soil after 2 weeks.

In all experiments seeds were harvested from wild-type and transgenic plants grown under identical conditions.

Seed Sterilization

?

Cloning of AtOGG1 and generation of AtOGG1 overexpression and RNA-interference-silenced lines

Obtaining cDNA Done!

The full-length cDNA sequence of AtOGG1 was obtained from GenBank (accession no. NM_102020).

Selecting Primers

?

Primer Pairs

"The primer pair 5#-ACGGCGATGAAGAGACCTC-3# and 5#-GATTCCTCGTAGCTTAGGTAGAC-3# was used for gene cloning."

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Using cDNAs generated from Arabidopsis leaves, a fragment containing the open reading frame (ORF) of AtOGG1 was amplified by PCR and subcloned into the pGEM T-Easy vector (Promega, Madison, WI, USA).

???

To construct the plant-transforming vector, the ORF of AtOGG1 was amplified using the primer pair 5#-TCCCCCGGGATGAAGAGACCTCGACCTAC-3# and 5#-CGAGCTCTCATGGCTTCAACGTATCAC-3#. The PCR products were digested by SmaI and SacI (indicated by underlining in the forward and reverse primers above) and subcloned into the binary plasmid pBI121 (Clontech, Palo Alto, CA, USA) by replacing the b-glucuronidase (GUS) gene, to generate the plant transforming vector pBI121-AtOGG1.

???

The plant-transformation vector containing AtOGG1 under the control of the cauliflower mosaic virus 35S promoter was electroporated into Agrobacterium tumefaciens strain EHA105. Transformation of A. thaliana was conducted by the floral dip method (Clough and Bent, 1998).

???

T0 seeds were harvested and germinated on a sterile medium containing 50 lg ml 1 kanamycin to select the transformants. The heterozygous transgenic plants were further characterized by a 3:1 segregation with respect to kanamycin resistance. Similarly, T3 homozygous seeds were obtained and confirmed by resistance.

???

To obtain AtOGG1-silenced lines, RNA interference (RNAi) vector pFGC5941 (TAIR) was used for generation of the silencing constructs. A 378 bp region of AtOGG1 was amplified by PCR to generate inverted repeat products using two primer pairs: 5#-CATGCCATGGATGGATGAAGAGACCTCGAC-3# and 5#-GGGATTTAAATGAAATCAGACCATAGCTCAG-3# (the first pair, forward and reverse), and 5#-TCCCCCGGGATGGATGAAGAGACCTCGAC-3# and 5#-CGGGATCCGAAATCAGACCATAGCTCAG-3# (the second pair, forward and reverse). The products were subcloned into pFGC5941 in a two-step cloning procedure that orients the fragments as inverted repeats separated by an intron from Petunia hybrida chalcone synthase A gene. The RNAi construct was then electroporated into strain EHA105 and used to transform Arabidopsis as described above. Transformed plants were selected by basta resistance. The transcript and protein levels of AtOGG1 in basta-resistant plants were determined by real-time PCR and Western blotting respectively.

Subcellular Localization Assay

Not doing.

RNA Extraction and Real-Time PCR

RNA Extraction

Total RNA was extracted from dry mature and imbibing seeds of transgenic and wild-type plants using the Universal Plant RNA Extraction Kit (BioTeke, Beijing, China).

RNA Purification

Purified RNA was digested with RNase-free DNase I (Takara, Dalian, China) to eliminate DNA contamination.

RNA Validation

RNA quality and quantity were determined by electrophoresis and spectrophotometry.

???

First-strand cDNA was synthesized from the total RNA with the PrimeScript 1st Strand cDNA Synthesis Kit (Takara).

Real-Time PCR

Real-time PCR was per formed on an IQ5 Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Green Real-time PCR Master Mix (Toyobo, Shanghai, China).

AtActin2 was used as an internal reference.

Gene-specific primers (forward and reverse, respectively): for AtOGG1 were 5#-TACAGAGCCAAATACATAACAG-3# and 5#-TGCTACCTTCGGACCAAC-3#; for AtMMH (AT1G52500) were 5#-AGCCAGAATCCACCCGTT-3# and 5#-GTCCTTCCACCAGCAGTAATG-3#; for AtARP (AT2G41460) were 5#-TATCAACAACAGCAAGCGAA-3# and 5#-TTCTTGAACAGTCTCGCCTC-3#; for AtLIG1 (AT1G08130) were 5#-GCGGTTAGGGTTCTCAGGT-3# and 5#-TCCACACACCGCCACTTAG-3#; for AtPARP2 (AT2G31320) were 5#-CGTATTCTGCGTCCTGTATTGT-3# and 5#-CGTCTCTGATATCTGTCAGTCCAC-3#; for AtRAD51 (AT5G20850) were 5#-TGAGGGAACATTCAGGCCAC-3# and 5#-AGAGAGCGGTAGCACTATCG-3#; and for AtActin2 (AT3G18780) were 5#-ATTACCCGATGGGCAAGTCA-3# and 5#-TGCTCATACGGTCAGCGATA-3#.

The conditions for real-time PCR were as follows: 95 C for 5 min, 40 thermal cycles of 95 C for 10 s, 58 C for 10 s, and 72 C for 20 s, followed by 75 thermal cycles with ramping at a rate of 0.5 C between 58 and 95 C to check fusion curves and verify the specificity of the PCR amplification.

The amount of cDNA was calculated using the comparative CT method (Schmittgen and Livak, 2008) using Bio-Rad iQ5 2.0 Standard Edition Optical System software. Data represent three biological replicates each consisting of three technical replicates.

Controlled Deterioration Treatment

(CDT)

???

CDT was conducted according to Tesnier et al. (2002) and Oge et al. (2008) with minor modifications. Dry mature seeds were harvested and stored at 4 C for 15 days before CDT. Each step of CDT was performed in airtight tube carriers containing appropriate saturated solution of salts to obtain stable relative humidity (RH). The carriers were placed in a dark incubator at appropriate temperature for various numbers of days. Temperature and RH were monitored with a Testo 610 controller placed inside the tube carriers. Seeds were placed in microcentrifuge tubes with lids removed and equilibrated for 3 days at 85% RH (15 C) in the presence of an appropriate saturated solution of KCl. Untreated controls were immediately dried for 3 days at 33% RH (20 C) in the presence of an appropriate saturated solution of MgCl2. For the controlled treatment, a saturated solution of KCl was used to obtain 82% RH at 40 C to equilibrate the seeds to 15–20% moisture content. Seeds were treated under these storage conditions for 1–7 days. After that, seeds were dried for 3 days at 33% RH (20 C).

Seed Sterilization

Then seeds were surface sterilized and stored at 4 C for 2 days before sowing.

Seed Sowing

Four sets of 100 seeds were used for each genotype and germination was monitored with a microscope every day and radicle protrusion of the seeds was scored until a plateau was reached, indicating completion of seed germination.

Cotyledon Expansion Growth of True Leaves

Cotyledon expansion was scored 10 days after sowing and was expressed as a percentage of the seeds that had germinated. Seed moisture content was measured by weighing the seeds before and after drying at 105 C for 24 h, and relative moisture content was expressed as percentage fresh weight for each RH. This study was replicated for three times and similar results were obtained.

Abiotic Stress Treatments

Chemical-Induced Stress

For chemical-induced stress treatments, four replicates of 100 dry mature seeds of each genotype were germinated in Petri dishes containing half-strength MS medium with 5–200 lM MV and 75 200 mM NaCl, or 150–600 mM mannitol (Man).

Thermotolerance Assay

The thermotolerance assay was conducted according to (Yokotani et al., 2008) with minor modifications. Briefly, four replicates of 100 dry mature seeds of each genotype were placed in microcentrifuge tubes with the lid removed and equilibrated for 3 days at 85% RH (15 C) before heat stress. After equilibration, the tubes were first immersed in water at 50, 51, and 52 C for 30 min, and then immediately dipped in water at room temperature to eliminate the heat stress. Seeds were surface sterilized and sown on half-strength MS medium. Germination and cotyledon expansion percentages were obtained as described for CDT.

DNA Extraction and Quantification of 8-oxo-dG

DNA Extractions

Total genomic DNA was extracted from seeds of each genotype with or without CDT using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Purified DNA was digested with proteinase K (10 mg ml�1, Sigma-Aldrich) for 2 h at 37 C according to the manufacturer’s instructions and subsequently deproteinized using chloroform/isoamyl alcohol. DNA (5 lg) was hydrolysed by in cubating with S1 nuclease (10 units; Takara) for 3 h at 37 C followed by treatment with alkaline phosphatase (10 units; Takara) for 2 h at 37 C. Free deoxynucleosides were isolated by filtering the whole reaction mixture using an Amicon Ultra-0.5 (Millipore, Billerica, MA, USA) filtration unit. Detection and quantification of 8-oxo-dG in the purified deoxynucleosides were carried out using the DNA Damage ELISA Kit (Stressgen, Enzo, San Diego, CA, USA) according to the manufacturer’s instructions.

Protein Expression and Western Blotting

Not doing. We're only looking at DNA/RNA.

Tetrazolium Assay

Not doing.

Raw Methodology

Plant Materials and Growth Conditions

A. thaliana (ecotype Columbia-0) plants were grown routinely in a greenhouse under 22±1 °C with a light/dark regime of 16 h light/8 h dark. Sterilized seeds were sown on Petri dishes containing half-strength Murashige and Skoog (MS) medium (Duchefa, Haarlem, The Netherlands) and then stratified at 4 C for 2 days. After stratification, seeds were germinated under the same growth conditions as above and seedlings were transferred to soil after 2 weeks. In all experiments seeds were harvested from wild-type and transgenic plants grown under identical conditions.

Cloning of AtOGG1 and generation of AtOGG1 overexpression and RNA-interference-silenced lines

The full-length cDNA sequence of AtOGG1 was obtained from GenBank (accession no. NM_102020). The primer pair 5#-ACGGCGATGAAGAGACCTC-3# and 5#-GATTCCTCGTAGCTTAGGTAGAC-3# was used for gene cloning. Using cDNAs generated from Arabidopsis leaves, a fragment containing the open reading frame (ORF) of AtOGG1 was amplified by PCR and subcloned into the pGEM T-Easy vector (Promega, Madison, WI, USA). To construct the plant-transforming vector, the ORF of AtOGG1 was amplified using the primer pair 5#-TCCCCCGGGATGAAGAGACCTCGACCTAC-3# and 5#-CGAGCTCTCATGGCTTCAACGTATCAC-3#. The PCR products were digested by SmaI and SacI (indicated by underlining in the forward and reverse primers above) and subcloned into the binary plasmid pBI121 (Clontech, Palo Alto, CA, USA) by replacing the b-glucuronidase (GUS) gene, to generate the plant transforming vector pBI121-AtOGG1. The plant-transformation vector containing AtOGG1 under the control of the cauliflower mosaic virus 35S promoter was electroporated into Agrobacterium tumefaciens strain EHA105. Transformation of A. thaliana was conducted by the floral dip method (Clough and Bent, 1998). T0 seeds were harvested and germinated on a sterile medium containing 50 lg ml 1 kanamycin to select the transformants. The heterozygous transgenic plants were further characterized by a 3:1 segregation with respect to kanamycin resistance. Similarly, T3 homozygous seeds were obtained and confirmed by resistance. To obtain AtOGG1-silenced lines, RNA interference (RNAi) vector pFGC5941 (TAIR) was used for generation of the silencing constructs. A 378 bp region of AtOGG1 was amplified by PCR to generate inverted repeat products using two primer pairs: 5#-CATGCCATGGATGGATGAAGAGACCTCGAC-3# and 5#-GGGATTTAAATGAAATCAGACCATAGCTCAG-3# (the first pair, forward and reverse), and 5#-TCCCCCGGGATGGATGAAGAGACCTCGAC-3# and 5#-CGGGATCCGAAATCAGACCATAGCTCAG-3# (the second pair, forward and reverse). The products were subcloned into pFGC5941 in a two-step cloning procedure that orients the fragments as inverted repeats separated by an intron from Petunia hybrida chalcone synthase A gene. The RNAi construct was then electroporated into strain EHA105 and used to transform Arabidopsis as described above. Transformed plants were selected by basta resistance. The transcript and protein levels of AtOGG1 in basta-resistant plants were determined by real-time PCR and Western blotting respectively.

Subcellular Localization Assay

To investigate the subcellular location of AtOGG1, vector pA7 YFP, a pre-made vector in pUC18 containing a YFP gene (Voelker et al., 2006), was used to study transient gene expression. Using the primer pair 5#-GCGTCGACATGAAGAGACCTCGACCTAC-3# and 5#-TCCCCCGGGTGGCTTCAACGTATCAC-3#, SalI and SmaI sites (indicated by underlining in the primer pair above) were introduced into the ORF of AtOGG1 and the resulting DNA fragment was cloned into pA7-YFP via the SalI and SmaI sites to generate the vector AtOGG1–YFP. The recombinant construct was electroporated into protoplasts of Araidopsis suspension-cultured cells. For the colocalization assay, Arabidopsis protoplasts were co-electroporated with AtOGG1 YFP, and the DNA of a single organelle marker as indicated. Mitotracker (Invitrogen, Carlsbad, CA, USA) was used to stain mitochondria. The subcellular locations of fusion proteins were examined by confocal laser scanning microscopy 12–16 h after electroporation. Transient expression analysis was carried out essentially as previously described (Miao and Jiang, 2007).

RNA Extraction and Real-Time PCR

Total RNA was extracted from dry mature and imbibing seeds of transgenic and wild-type plants using the Universal Plant RNA Extraction Kit (BioTeke, Beijing, China). Purified RNA was digested with RNase-free DNase I (Takara, Dalian, China) to eliminate DNA contamination. RNA quality and quantity were determined by electrophoresis and spectrophotometry. First-strand cDNA was synthesized from the total RNA with the PrimeScript 1st Strand cDNA Synthesis Kit (Takara). Real-time PCR was performed on an IQ5 Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the SYBR Green Real-time PCR Master Mix (Toyobo, Shanghai, China). AtActin2 was used as an internal reference. Gene-specific primers (forward and reverse, respectively): for AtOGG1 were 5#-TACAGAGCCAAATACATAACAG-3# and 5#-TGCTACCTTCGGACCAAC-3#; for AtMMH (AT1G52500) were 5#-AGCCAGAATCCACCCGTT-3# and 5#-GTCCTTCCACCAGCAGTAATG-3#; for AtARP (AT2G41460) were 5#-TATCAACAACAGCAAGCGAA-3# and 5#-TTCTTGAACAGTCTCGCCTC-3#; for AtLIG1 (AT1G08130) were 5#-GCGGTTAGGGTTCTCAGGT-3# and 5#-TCCACACACCGCCACTTAG-3#; for AtPARP2 (AT2G31320) were 5#-CGTATTCTGCGTCCTGTATTGT-3# and 5#-CGTCTCTGATATCTGTCAGTCCAC-3#; for AtRAD51 (AT5G20850) were 5#-TGAGGGAACATTCAGGCCAC-3# and 5#-AGAGAGCGGTAGCACTATCG-3#; and for AtActin2 (AT3G18780) were 5#-ATTACCCGATGGGCAAGTCA-3# and 5#-TGCTCATACGGTCAGCGATA-3#. The conditions for real-time PCR were as follows: 95 C for 5 min, 40 thermal cycles of 95 C for 10 s, 58 C for 10 s, and 72 C for 20 s, followed by 75 thermal cycles with ramping at a rate of 0.5 C between 58 and 95 C to check fusion curves and verify the specificity of the PCR amplification. The amount of cDNA was calculated using the comparative CT method (Schmittgen and Livak, 2008) using Bio-Rad iQ5 2.0 Standard Edition Optical System software. Data represent three biological replicates each consisting of three technical replicates.

Controlled Deterioration Treatment

(CDT)

CDT was conducted according to Tesnier et al. (2002) and Oge et al. (2008) with minor modifications. Dry mature seeds were harvested and stored at 4 C for 15 days before CDT. Each step of CDT was performed in airtight tube carriers containing appropriate saturated solution of salts to obtain stable relative humidity (RH). The carriers were placed in a dark incubator at appropriate temperature for various numbers of days. Temperature and RH were monitored with a Testo 610 controller placed inside the tube carriers. Seeds were placed in microcentrifuge tubes with lids removed and equilibrated for 3 days at 85% RH (15 C) in the presence of an appropriate saturated solution of KCl. Untreated controls were immediately dried for 3 days at 33% RH (20 C) in the presence of an appropriate saturated solution of MgCl2. For the controlled treatment, a saturated solution of KCl was used to obtain 82% RH at 40 C to equilibrate the seeds to 15–20% moisture content. Seeds were treated under these storage conditions for 1–7 days. After that, seeds were dried for 3 days at 33% RH (20 C). Then seeds were surface sterilized and stored at 4 C for 2 days before sowing. Four sets of 100 seeds were used for each genotype and germination was monitored with a microscope every day and radicle protrusion of the seeds was scored until a plateau was reached, indicating completion of seed germination. Cotyledon expansion was scored 10 days after sowing and was expressed as a percentage of the seeds that had germinated. Seed moisture content was measured by weighing the seeds before and after drying at 105 C for 24 h, and relative moisture content was expressed as percentage fresh weight for each RH. This study was replicated for three times and similar results were obtained.

Abiotic Stress Treatments

For chemical-induced stress treatments, four replicates of 100 dry mature seeds of each genotype were germinated in Petri dishes containing half-strength MS medium with 5–200 lM MV and 75 200 mM NaCl, or 150–600 mM mannitol (Man). The thermotolerance assay was conducted according to (Yokotani et al., 2008) with minor modifications. Briefly, four replicates of 100 dry mature seeds of each genotype were placed in microcentrifuge tubes with the lid removed and equilibrated for 3 days at 85% RH (15 C) before heat stress. After equilibration, the tubes were first immersed in water at 50, 51, and 52 C for 30 min, and then immediately dipped in water at room temperature to eliminate the heat stress. Seeds were surface sterilized and sown on half-strength MS medium. Germination and cotyledon expansion percentages were obtained as described for CDT.

DNA Extraction and Quantification of 8-oxo-dG

Total genomic DNA was extracted from seeds of each genotype with or without CDT using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Purified DNA was digested with proteinase K (10 mg ml�1, Sigma-Aldrich) for 2 h at 37 C according to the manufacturer’s instructions and subsequently deproteinized using chloroform/isoamyl alcohol. DNA (5 lg) was hydrolysed by incubating with S1 nuclease (10 units; Takara) for 3 h at 37 C followed by treatment with alkaline phosphatase (10 units; Takara) for 2 h at 37 C. Free deoxynucleosides were isolated by filtering the whole reaction mixture using an Amicon Ultra-0.5 (Millipore, Billerica, MA, USA) filtration unit. Detection and quantification of 8-oxo-dG in the purified deoxynucleosides were carried out using the DNA Damage ELISA Kit (Stressgen, Enzo, San Diego, CA, USA) according to the manufacturer’s instructions.

Protein Expression and Western Blotting

The full-length cDNA of AtOGG1 was cloned into the pET14b vector (Novagen, San Diego, CA, USA) and expressed in E. coli strain BL21 (DE3) as described by Chu et al. (2011). The recombinant proteins were purified following the manufacturer’s protocol (Novagen). Polyclonal antiserum was raised in mouse and used in Western blotting. Western blot analysis was performed as described by Chu et al. (2011) with minor modifications. Protein samples were separated by SDS/PAGE and the gels were transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). The primary antibody was the polyclonal antiserum described above at 1:2000 dilution, and the secondary antibody was goat anti-mouse immunoglobulin horseradish peroxidase antibody (Sigma-Aldrich, St Louis, MO, USA) at 1:5000 dilution. ECL-Plus Western Blotting Detection Reagents (Invitrogen) were used for detection, according to the supplier’s instructions.

Tetrazolium Assay

Seed viability was estimated using tetrazolium staining. Arabidopsis seeds were incubated in a 1% (w/v) aqueous solution of 2,3,5 triphenyltetrazolium chloride (Alfa, Ward Hill, MA, USA) at 30 C in darkness for 2 days as described by Wharton (1955). Tetrazolium salts were metabolically reduced to highly coloured end products called formazans by NADH-dependent reductases of the endoplasmic reticulum