Basic HTML Version
Plant Gene and Trait, 2013, Vol.4, No.6, 30
-
36
http://pgt.sophiapublisher.com
33
Note: M: Molecular Weight Marker; A:
MsC4H
; B:
MsCAD
1.4 Sequencing results and sequences analysis
Sequencing results demonstrated that the length
of
MsC4H
and
MsCAD
fragment was 305 and 269
base pairs, which encoded 101 and 51 amino acids
(Figure 4 and Figure 5), respectively. Blastn analysis
revealed that the nucleotide sequences of
MsC4H
and
MsCAD
were highly homologous to many
C4H
and
CAD
cDNAs cloned from different species. The
homology
of
MsC4H
with
C4H
isolated
from
Sorghum
bicolor
(XM_002451999) and
Zea
mays
(NM_001157893) was up to 98% and 94%. The
homology of
MsCAD
with CAD cloned from
Brachypodium distachyon was 80%. BlastP analysis
revealed that the amino acid sequences encoding
by
MsC4H
and
MsCAD
had high homology with
C4H
and
CAD
protein from many plants. The homology of
C4H with C4H protein from
Zea mays
(NP_00115
1365) and
Oryza sativa
Japonica Group (NP_
001046814, NP_001046819) were uo to 90%. The
homology of
CAD
with CAD from
Zea mays
(NP_
001131273, DAA35929) was more than 83%.
Moreover, C4H protein contained CyPX (Cytochrome
P450 monooxygenases) superfamily conserved
domain, and CAD protein contained MDR (Medium-
chain dehydrogenase/reductase) superfamily NAD(P)
binding sites, which indicated the isolated genes in
this experiment were true
C4H
and
CAD
genes. The
sequences of
MsC4H
and
MsCAD
had been submitted
to Genbank, whose accession numbers were
JQ598686 and JQ598683.
2 Discussion
C4H was involved in the initiation reaction of
biosynthesis of lignin monomer, which was the second
enzyme in lignin metabolism pathway. Under the
action of phenylalanine ammonia lyase (PAL),
phenylalanine was deaminated to cinnamic acid, and
then was converted to 4-coumaraic acid by C4H. After
the methylation reaction and subsequent ligation
reaction, three kinds of hydroxy cinnamic acid CoA
esters were generated. The latest research
demonstrated that saccharification efficency of the
secondary wall of the antisense
C4H
transgenic
tobacco was improved (Cook and Daudi, 2012), hence
providing the new strategy and basis to improve the
applications of the biomass crops in the industry.
CAD is the last enzyme in lignin monomer
biosynthesis, involving the reduction reaction with
Cinnamoyl-CoA reductase (CCR). CCR restores three
kinds of hydroxy cinnamic acid CoA esters to form
the corresponding cinnamic aldehyde, and then CAD
deoxidized it to three kinds of cinnamyl alcohol. The
enzyme activity of CAD of antisense
CAD
transgenic
Figure 4 Nucelotide sequence of
C4H
and the encoded amino acids sequence of
Miscanthus sinensis