Descripción y análisis de los guiones implementados para la MD

High throughput DNA was isolated by a Nimbus liquid handing robot. Leaf tissue (5 cm lengths) was collected and placed into a deep-well microtiter plate. The plate was frozen at -80 °C for 30 min and frozen dry overnight. One SDS and ethanol washed stainless steel ball bearing was added to each well. The plate was sealed with 8-lid strips and shaken on the QIAGEN shaker at a frequency of 23/second for 2 min each side. The plate was spun by a centrifuge for 15 min at 3,000 rpm to settle contents before opening the lids, then 375 μl extraction buffer (0.1 M Tris-HCl, pH 8.0; 0.05 M EDTA, pH 8.0 and 1.25% SDS, pre- warmed to 65 °C) was added to each well and incubated at 65 °C for one hour in an oven. The plate was cooled in a fridge to room temperature and 187 μl 6 M ammonium acetate (stored at 4 °C) was added to each well. The lysate was mixed by inverting the plate and left in the fridge for 30 min. The plate was centrifuged for 30 min at 3000 rpm and 340 μl supernatant was recovered into a new deep well microtiter plant containing 220 μl isopropanol. DNA was allowed to precipitate for 5 min at room temperature. The plate was centrifuged for 30 min at 3,000 rpm and the supernatant was poured off. Then 70% ethanol (320 μl) was added to the plate and centrifuged for 30 min at 3,000 rpm. Supernatant was poured off and DNA was resuspended in 225 μl dH2O and left overnight

at 4 °C for the DNA pellet to dissolve. The plate was centrifuged for 20 min at 3,000 rpm and 150 μl of supernatant was transferred to a new microtiter plate. The DNA was ready to use.

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Table 2.1 Primers used in the experiments

Primer No. Details Sequence

PRR-101 Fwd: Amplify PCR fragment

for expression of OsALMT1 5’-GTATAAGGATTGACATTTGCC-3’ PRR-102 Rev: Amplify PCR fragment

for expression of OsALMT1 5’-GCCACCTGAATAACAACTAC-3’ PRR-103 Fwd: Amplify PCR fragment

for expression of OsALMT1 5’-GTTCTTCGCCACTGTGCCT-3’ PRR-104 Rev: Amplify PCR fragment

for expression of OsALMT1 5’-GAATGTTGCAGACGCTCG-3’ PRR-105 Fwd: Amplify PCR fragment

for expression of OsALMT2 5’-GAGGCGATGACCGAAGCGAGC-3’ PRR-106 Rev: Amplify PCR fragment

for expression of OsALMT2 5’-CAACTCCGGCCACCATGCTC-3’ PRR-107 Fwd: Amplify PCR fragment

for expression of OsALMT6 5’-TCGCGGGAGAGGAGGAGG-3’ PRR-108 Rev: Amplify PCR fragment

for expression of OsALMT6 5’-GATCGCTCACGGTTTGCAG-3’ PRR-109 Fwd: Amplify PCR fragment

for expression of OsALMT3 5’-CGTCATCAGAGGCAGAGCAGC-3’ PRR-110 Rev: Amplify PCR fragment

for expression of OsALMT3 5’-GTTGGAGTTGGGTACGGGCA-3’ PRR-111 Fwd: Amplify PCR fragment

for expression of OsALMT8 5’-GGTGGGACAGCTGGTGAAG-3’ PRR-112 Rev: Amplify PCR fragment

for expression of OsALMT8 5’- GCGTGGGCCGTGAAGTCC-3’ PRR-113 Fwd: Amplify PCR fragment

for expression of OsALMT7 5’- GGTGGGTGGAAGGAGGGC-3’ PRR-114 Rev: Amplify PCR fragment

for expression of OsALMT7 5’- CTTCCTCCAGCTTTCCCAG-3’ PRR-115 Fwd: Amplify PCR fragment

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Primer No. Details Sequence

PRR-116 Rev: Amplify PCR fragment

for expression of OsALMT4 5’- GCTCTCGTAGGTTCTCGCC-3’ PRR-117 Fwd: Amplify whole length

cDNA for OsALMT1 5’-GCGGATCCATGAATGGAAAGAAGGGT-3’ PRR-118 Fwd: Amplify whole length

cDNA for OsALMT1 5’-GGGGTACCATGAATGGAAAGAAGGG-3’ PRR-119 Rev: Amplify whole length

cDNA for OsALMT1 5’-CCGGAATTCTTAGTTTGCAATCTGCTG-3’ PRR-120 pUC/M13 Forward Primer 5´-CGCCAGGGTTTTCCCAGTCACGAC-3´ PRR-121 pUC/M13 Reverse Primer 5´-TCACACAGGAAACAGCTATGAC-3´ PRR-122 Fwd: Amplify OsALMT1

Promoter 5’-TGCCCGGGACCTGTTACTACTTGTTATGC-3’ PRR-123 Rev: Amplify OsALMT1

Promoter 5’-CTGGGTACCTCTCTAACTTGCGGTCTCTT-3’ PRR124 Middle primer for OsALMT1

promoter sequencing 5’-GTTCCTACTTTGGGTGCGACAT-3’ PRR125 Middle primer for OsALMT1

promoter sequencing 5’AGTTTTGTGATTCCTCTGGT-3’ PRR-128 Fwd: primer for OsALMT1

Pro location 5’-CCTTAATTAAACCTGTTACTACTTGTTATGC-3’ PRR-129 Rev: primer for OsALMT1

Pro location 5’-TTGGCGCGCCTCTCTAACTTGCGGTCTCTT-3’ PRR-130 Reverse primer for

ALMT1::GFP

5’-GGTGAACAGCTCCTCGCCCTTGCTCACCAT GTTTGCAATCTGCTGCTTGAAC-3’

PRR-131 Forward primer for ALMT1::GFP

5’- TGGCAAGGTTCAAGCAGCAGATTGCAAAC ATGGTGAGCAAGGGCGAGGAGCTGTTCACC-3’ PRR-132 Reverse primer for GFP 5’-CGGAATTCTTACTTGTACAGCTCGTC-3’ PRR-133 Forward primer for GFP 5’-GCGGATCCATGGTGAGCAAGGGCGAGG-3’

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Primer No. Details Sequence

PRR-134 Reverse primer for GFP::ALMT1

5’-TCCTTATACTACCCTTCTTTCCATTCAT CTTGTACAGCTCGTCCATGCCG-3’

PRR-135 Forward primer for GFP::ALMT1

5’-ATCACTCACGGCATGGACGAGCTGTACAAG ATGAATGGAAAGAAGGGTAGT-3’

PRR-136 Forward primer for

OsALMT1 RNAi 5’-GGATCCGGTACCCAAACTAGCTTAC-3’ PRR-137 Reverse primer for OsALMT1

RNAi 5'- CCCGGGACTAGTCATCATCTCATGG -3' PRR-138 Forward primer for pART-

OsALMT1::GFP 5’-GAATTCATGAATGGAAAGAAGGGT-3’ PRR-139 Reverse primer for pART-

OsALMT1::GFP 5’-GGATCCTTACTTGTACAGCTCGTC-3’ PRR-140 Forward primer for pART-

GFP::OsALMT1 5’-GAATTCATGGTGAGCAAGGGCGAG-3’ PRR-141 Reverse primer for pART-

GFP::OsALMT1 5’-GGATCCTTAGTTTGCAATCTGCT-3’ PRR-142 Forward primer for

OsALMT1 qRT-PCR 5’-CCTTAGAAGAGTGTGTCAAGAAG-3’ PRR-143 Reverse primer for OsALMT1

qRT-PCR 5’-CCATTTAGCAGAGTTCGCCAG-3’ PRR-80F Forward primer for genotype

identification 5’-TGCAGCATCTATTCATATGCTCT-3’ PRR-80R Reverse primer for genotype

identification 5’-AACACCAAACAACAGGGTGAG-3’ UbiP Forward primer for

sequencing pUbi-OsALMT1 5’-CTTGATATACTTGGATGATGG-3’ TM/pWUbi Reverse primer for sequencing

pUbi-OsALMT1 5’-GTGTTCTAAGCTAGCCTGG-3’ 35SP Forward primer on 35S

promoter 5’-TATCCTTCGCAAGACCCTTCCT-3’ TM ocs Reverse primer on ocs

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Primer No. Details Sequence

OsGAPDHF Forward primer for qRT-PCR

as a reference 5’-GTTGAGGGTTTGATGACCAC-3’ OsGAPDHR Reverse primer for qRT-PCR

as a reference 5’-TCAGACTCCTCCTTGATAGC-3’ OseEF-1α F Forward primer for qRT-PCR

as a reference 5’-TTTCACTCTTGGTGTGAAGCAGAT-3’ OseEF-1α R Reverse primer for qRT-PCR

as a reference 5’-GACTTCCTTCACGATTTCATCGTAA-3’ Oligo dT Primer for cDNA synthesis 5’-TTTTTTTTTTTTTTTTTTTTVN-3’

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2.4.3 RNA extraction and 1

strand cDNA synthesis

Total RNA from leaf and root tissues were extracted by using the RNeasy® Plant Mini Kit, QIAGEN. Residual DNA was removed by on column DNase digestion by using the RNase-Free DNase Set, QIAGEN. Leaf tissue (~100 mg) was put into 1.5 ml Eppendorf tube and frozen in liquid nitrogen. The sample was grounded into a powder with a sterile plastic stick. Buffer RLT (450 μl with 40 μl/mL 1 M Dithiothreitol (DTT)) was added to the powdered tissue and vortexed vigorously. The lysate was transferred to a QIAshredder spin column placed in a 2 ml collection tube, and centrifuged for 2 min at full speed. The supernatant of the flow-through was carefully transferred to a new microcentrifuge tube without disturbing the cell-debris pellet in the collection tube. Ethanol (96–100%) of 0.5 volume of the flow-through was added to the cleared lysate and mixed immediately by pipetting. The sample, including any precipitate that may have formed, was transferred to an RNeasy spin column placed in a 2 ml collection tube. The lid was gently closed, and centrifuged for 15 s at 10,000 rpm with the flow-through discarded. Buffer RW1 (350 μl) was added to the RNeasy spin column. The lid was gently closed, and centrifuged for 15 s at 10,000 rpm to wash the spin column membrane with the flow-through discarded. The DNase I incubation mix (10 μl DNase I stock solution to 70 μl Buffer RDD) was directly added to the RNeasy spin column membrane, and placed on the benchtop for 15 min at room temperature. Buffer RW1 (350 μl) was added to the RNeasy spin column. The lid was gently closed, and centrifuged for 15 s at 10,000 rpm with the flow-through discarded. Buffer RPE (500 μl) was added to the RNeasy spin column. The lid was gently closed, and centrifuged for 15 s at 10,000 rpm to wash the spin column membrane with the flow- through discarded. Another 500 μl Buffer RPE was added to the RNeasy spin column and repeat the centrifuge steps. The RNeasy spin column was placed in a new 2 ml collection tube, and the old collection tube was discarded with the flow-through. The lid was gently closed, and centrifuged at full speed for 1 min. The RNeasy spin column was placed in a new 1.5 ml collection tube and 50 μl RNase-free water was directly added to the spin column membrane. The lid was gently closed, and centrifuged for 1 min at 10,000 rpm to elute the RNA. The RNA was ready for next step.

First strand cDNA was synthesised by SuperScript® III Reverse Transcriptase, InvitrogenTM_.

Total RNA (1 μg) and oligo(dT)20 (1 μl, 0.5 μg/μl) were added to a nuclease-free

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min and incubated on ice for at least 1 minute. The content was collected by brief centrifugation and 4 μl 5X First-Strand Buffer, 2 μl 0.1 M DTT, 1 μl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH) and 0.5 μl of SuperScript™ III RT (200 units/μl) were added. The content was immediately incubated at 42 °C for one hour in a PCR machine. RNase H (0.25 μl, 4 U/μl) was added to the tube and incubated at 37 °C for 30 min to remove the redundant RNA.

2.5 PCR reactions