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Table 2 Primers used in experiment
Usage
Primer name
Oligonucleotide sequence (5’–3’)
KR-F
ATGGCTTCTCTCTCCGCTTC
KR-R
TTACATCACCATACCTCCATCAATG
HD-F
ATGGCAGCCTCTGCTCTCTC
HD-R
CTATTCACTCCCTCCCGAGC
ENR-F
ATGGCAACAACACCGTTTTCT
Full-length cDNA sequence cloning
ENR-R
CTAATGCTGGTCCTTGGGAATG
qActin-F
TTGGAATGGGTCAGAAGGATGC
qActin-R
AGTGGTGCCTCAGTAAGAAGC
qKR-F
GAACTGCCAACTTCTCCTCCTC
qKR-R
CTGCCTCCTCAAGCGTAGC
qHD-F
CCTCTGATTCCGATGCTGCTC
qHD-R
TGCGGGATACCTTAGTTCAATGG
qENR-F
AGACTTCGGCACCATAGACATC
Real-time RT-PCR
qENR-R
GCGGATAAAGCAGCAAGATACC
3.5 Quantitative real-time RT-PCR
The real-time RT-PCR analysis was performed by
using a LightCycler 2.0 instrument system (Roche,
Germany). β
-
actin gene was taken as reference gene.
Three pairs of gene-specific primers (Table 1) were
designed according to the AhKR, AhHD and AhENR
gene sequences. The real-time RT-PCR reactions were
performed by using the SYBR Premix Ex
Taq
polymerase (TaKaRa, Japan) according to the
manufacturer’s instructions. The expression of the
gene was calculated relative to the calibration sample
and the β
-
actin to normalize the sample input amount.
All the experiments were performed in triplicate to
ensure the data accuracy.
Acknowledgements
This work was financed by the earmarked fund for Modern
Agro-industry Technology Research System (nycytx
-
19),
National High-Tech Research and Development Plan of China
(No.2006AA10A114 and 2007AA10Z189), National Project of
Scientific and Technical Supporting Program (2008BAD97B04)
and the Natural Science Fund of Shangdong Province
(ZR2009DQ004)
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