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Plant Gene and Trait, 2013, Vol.4, No.6, 30
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34
Figure 5 Nucelotide sequence of
CAD
and the encoded amino acids sequence of
Miscanthus sinensis
Medicago sativa
L. was reduced dramatically.
Although the lignin content remains unchanged, but
the content of the S monomer decreased, resulting in
S/G (syringyl/guaiacyl) ratio and S+G yield was
significantly reduced. The digestibility of the
genetically modified alfalfa by
CAD
gene has been
improved. The enzyme activity of transgenic
Populus
tremula
×
Populus
alba
of
antisense
and
sense
CAD
decreased 70% compared to the control,
hence lowering the use of the chemicals to get rid of
the lignin during the pulp and paper process and
reducing the cost of pulp and paper.
Based on two pairs of the specific PCR primers,
cDNA fragments of
C4H
and
CAD
were isolated from
bioenergy plant,
Miscanthus
, therfby laying a
foundation to clone the full-length cDNA sequences
of
C4H
and
CAD
by RACE technique in the future.
We also cloned the cDNA fragments encoding
caffeoyl CoA 3-O-methyltransferase (CcoAOMT)
(JQ598685) and peroxidase (POD) (JQ598684)
from
Miscanthus
. We plan to carry out quantitative
gene expression analysis of these four genes by qPCR
technique, and analyze the correlation of mRNA
transcription expression level of these genes with the
saccharification efficiency, therefore providing the
transformation target gene in order to orientedly
molecularly improve the saccharification efficiency of
the
Miscanthus
germplasm.
3 Materials and methods
3.1 Materials
3.1.1 Plant material
The first internode of
Miscanthus
, plant material used
in this experiment, was collected and frozen in liquid
nitrogen, and then was taken to the laboratory and was
immediately placed in -80
℃
refrigerator.
3.1.2 Strain and vector
Escherichia coli
strain DH5α was kept in our own
laboratory. Cloning vector pMD18-T was bought from
TaKaRa Biotechnology (Dalian) Co. LTD.
3.1.3 Reagent
Dnase I, M-MLV RTase cDNASynthesis Kit,
Taq
DNA
polymerase and Agarose Gel DNA Purification Kit
were purchased from TaKaRa Biotechnology (Dalian)
Co. LTD. Other biochemical reagents were all
analytical grade reagents imported or domestically
produced.
3.2 Methods
3.2.1 Total RNA extraction
The total RNA was isolated from
Miscanthus
according
to the modified CTAB-LiCl method (Gasic et al.,
2004). After the total RNA was digested by Dnase I to
get rid of the residual trace DNA contamination, it was
characterized by 1.2% agarose gel electrophoresis.
3.2.2 Primers design
Based on
C4H
and
CAD
sequences of several monocotes
reported in Genbank, two pairs of PCR primers
C4H-F (5'-CTCGCGCAGAGCTTCGACTA), C4H-R
(5'-TCCCACTCGATGGACCAGAG) and CAD-F
(5'-CGGAAGGAATCGACACAGAG), CAD-R (5'-G
CACCAACCTGAGTTACAAC), were designed with
Primer Premier 5.0 software. All the primers were
synthesized by Sangon Biotech (Shanghai) Co. Ltd.
3.2.3 cDNA synthesis
cDNA was synthesized according to the protocol of
M-MLV RTase cDNA Synthesis Kit, and then was
placed in -20
℃
refrigerator.
3.2.4 RT-PCR amplification
50 µL PCR reaction system was composed of 5 µL
10×PCR Buffer, 5 µL 2.0 mmol/L each dNTP, 1 µL
cDNA, 1.5 µL 10 pmol/µL forward and reverse primer,
respectively, 1 µL
Taq
DNA polymerase and 35 µL
ddH
2
O. Thermocycling was performed at 95
℃
for 5 min,
then at 95
℃
for 30 s, 54
℃
for 30 s, 72
℃
for 1 min
for 35 cycles, 72
℃
for 7 min. The PCR products
were detected by 1% Agarose gel electrophoresis.
3.2.5 Characterization of the positive clones
The target PCR products were recovered with
TaKaRa
TM
Agarose Gel DNA Purification Kit. Then
the purified PCR products were ligated to pMD18-T