14 - GAB-2015v6n1页

Bioscience Methods 2014, Vol.6, No.1, 1-13
http://bm.biopublisher.ca
11
Reverse transcription of mRNA was performed from
the total RNA using a Prime Script™ RT reagent Kit
(TaKaRa Biotechnology, Dalian, China). The
construction and screening of a subtractive library was
performed as described previously (Ma et al.2011).
Based on BLAST analysis with the ESTs, a StOSM
specific primer, O-F/O-R, was designed and
synthesised. Using the primers O-F (5’-ATGGG
CTACTTGAGATCTTC3’) and O-R (5’-TTACTTGG
CCACTTCATCAG-3’), the general protocol of
molecular cloning was followed to clone the
full-length StOSM cDNA.
3.3 Phylogenetic and gene structure analysis
The sequence of the cloned StOSM cDNA was
compared against the NCBI files and the
PGSC_DM_v3.4_gene.fasta files from the Potato
Genome Sequencing Consortium (PGSC) website
(http://potatogenomics.plantbiology.msu.edu) (Xu
et al. 2011 and Cory et al. 2014). Using the
downloaded sequences for the StOSM and promoter
from the PGSC database, two phylogenetic trees for
the StOSM peptides and promoters were
constructed using the neighbour-joining method in
the MEGA 5.02 software package developed by
(Saitou and Nei1987 and Tamura et al. 2007).
Briefly, copy sequences in FASTA format to the
Alignment Explorer in the MEGA 5.02. Align
sequences by Clustal W. Import and save alignment
file in MEGA format. Construct Neighbor Joining tree
with 1000 Bootstrap Replications. The Genomic
distribution of the StOSM genes throughout the potato
chromosomes was drawn in proportion to their
positions in the PTGS (Release: Potato Genome
Brower v4.03). The selected StOSM expression on
FPKM values was compared with data downloaded
from the RH genotypes in the Potato Genome
Sequencing Consortium website (http://potatogenome.net),
Annotation report v3.4. RNA-Seq Gene Expression
Data. DM_RH_RNA-Seq_FPKM_ expression_
matrix.
3.4 Reverse transcription PCR for StOSM
expression analysis
A total of 300–350 ng of RNA was extracted from
leaves at WCM concentrations of 70%, 60%, 50%,
40%, 30%, 20% and 10%. Following the instructions
for Premix Ex TaqW Version 2.0 (TaKaRa), reverse
transcription PCR was performed using primers
(Table 3) designed specifically for each of the eleven
StOSMs. The PCR product amplified from the
treatment templates using beta-actin-specific primers
was used as an internal control.
Table 3 StOSM member -specific primers for reverse transcription PCR
StOSM- Primer Primer sequence Product (bp)
1G F 5'TGCCCCGACCAATCCTAG 3' 183
R 5'GAAAAATCTCGACCAGTCAGTAGG 3'
2D F 5'TGAATCAATTTAGCAACTTAGA 3' 131
R 5'CATTTATATTGGCAACACATT 3'
3B F 5'TTCTCCCTTCTTGCTTTTGTGACT 3' 150
R 5'CCCTTGGAGCATTGATGACC 3'
3C F 5'ATACGCTTTGAACCAGTTTG 3' 129
R 5'TTGGCTGTGCATTGAATT 3'
3F F 5'TTCTTCCTCCTTGCTTTTGTGAC 3' 149
R 5'CCTCGGCGCATTGATAACC 3'
5A F 5'TTCTCCCTTCTTGCTTTTGTGAC 3' 144
R 5'GCGCATCAATAACCCACGT 3'
8E F 5'CGCCCCGACCAATCCTA 3' 154
R 5'TGAAAAATCTGGACAAGTCGTTAG 3'
182 F 5'GGTGCCCTAATGCGTATA 3' 183
R 5'CGGGGTCGTGCCTATA 3'
251 F 5'ACAAGCCACCTACCCTAAT 3' 134
R 5'CGGTGGGTAAGTGAGTGA 3'
297 F 5'CGTCCATCATTCTATTCAAG 3' 147
R 5'CGTTGGGCCTCTTCAC 3'
251 F 5'AACTTGTCAACCATCTTTTTAC 3' 159
R 5'TTCTTCATTAGGCCTCTTTAC 3'