Page 11 - Genomics and Applied Biology

Genomics and Applied Biology, 2011, Vol.2 No.1
http://gab.sophiapublisher.com
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Lolium perenne L. EF495352, Zea mays BT016732 at
amino acid level by DNAStar software. On the other
hand, we assayed the protein transmembrane structure
of Put Px on line (http://www.cbs.dtu.dk/cgi-bin/nph)
by ProtParam soft. At last, the subcellular localization
of PutAPx gene was analysed and predicted
respectively by PSORT and ProtComp Version 611
softwares (http://www.softberry.Compberry.phtml).
3.6 Congstruction of yeast expression vector
pYES2-PutAPx and its antioxidation analysis
transforming into yeast
PCR the recombination plasmid pT-PutA Px and add
two restriction enzyme sites of BamHⅠand XbaⅠ to
ORF of pT-PutA Px, and then isolate and recover
pYES2 vector and target fragment after digesting by
BamHⅠ and XbaⅠ, then linke the two fragments by
T4 ligase to transforme into Escherichia coli JM109.
After identifing by the above enzymes, we assigned
the recombination plasmid as pYES2-PutAPx.
the pYES2-PutAPx was tranformed into yeast strain
INVScI by lithium acetate (Goetz et al., 1995), and
the clones were detected by PCR (F2: 5'-GGATCCAT
GGCGGCCCCGGT-3'; R2: 5'-TCTAGATTACTTGC
TCCTCTTG-3'). The positive clones were cultivated
on the SD medium. Diluted the yeast transformants
pYES2-PutAPx and pYES2 into 100 different
concentration, and cultivated on the SD medium with
0 mmol/L, 2 mmol/L, 4 mmol/L, 8 mmol/L, 16 mmol/L
H2O2 respectively. the OD600 was 2 and the yeast
solution was dilute 10-1, 10 -2, 10 -3, 10-4 respectively,
and were cultivated at 30℃ to observe the growth
status directly. Finally, comparing the adaptive
capability of recombination transformed yeasts under
oxidative stress.
3.7 Tissue specific expression analysis of PutA Px
The tested mateials about leaves, roots, stems, sheaths,
anthers, female flowers, scapes, pollinated ears of
Puccinellia tenuiflora were collected at flowering
phase and total RNA of them were extracted
respectively using TRizol. And then cDNA were
obtained by reverse transcription kit. PCR cDNA by
two specific primers (F3: 5'- CGATGGCGGCCCCG
GTGGTG-3', R3: 5'-CCTTACTTGCTCCTCTTGGA-3',
annealed at 56℃, 30 cycles) to analyse the Tissue
specific expression of PutA Px in different tissues via
detecting with 0.8% agarose gel electrophoresis.
Author Contributions
Qingjie Guan and Shenkui Liu conceived and conducted this
research and prepared the manuscript. Qingjie Guan, Lin Li and
Takano Tetsuo involved this research and collected data. All of
them had read the final version of this paper and agreed with
the authors’ credits.
Acknowledgements
This work was initiated by the Youth Science Fund of
Northeast Forestry University (07049) and Harbin Youth
Science and Technology Innovation Fund Project
(RC2007QN002079). We also thank two anonymous reviewers
for their strict criticism on this paper.
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