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Plant Gene and Trait, 2013, Vol.4, No.6, 30
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36
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30
Research Report Open Access
Cloning and Analysis of Gene Fragments Encoding
C4H
and
CAD
from
Miscanthus sinensis
Bingyou Fan , Fang Li
College of Agriculture, Henan University of Science and Technology, Luoyang, 471003,China;
Correspondings author, fanbingyou2005@163.com;
Authors
Plant Gene and Trait, 2013, Vol.4, No.6 doi: 10.5376/pgt.2013.04.0006
Received: 17 Jul., 2013
Accepted: 19 Jul., 2013
Published: 23 Jul., 2013
Copyright
© 2013 Fan and Li, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article
:
Fan and Li, 2013, Cloning and Analysis of Gene Fragments Encoding
C4H
and
CAD
from
Miscanthus sinensis
, Plant Gene and Trait, Vol.4, No.6 30-36 (doi:
10.5376/pgt.2013.04.0006)
Abstract
Based on cDNA sequences encoding Cinnamate-4-hydroxylase (C4H) and Cinnamoyl alcohol dehydrogenase (CAD)
isolated from several monocots reported in GenBank, two pairs of PCR primers were designed with Primer Premier 5.0 software. The
total RNA was extracted from
Miscanthus sinensis
according to CTAB-LiCl method and then cDNA was synthesized by reverse
transcription. PCR products, which were named as
MsC4H
and
MsCAD
, were successfully obtained by reverse transcription
polymerase chain reaction (RT-PCR) and then cloned into vector pMD18-T. The positive clones identified by plasmid PCR were
sequenced. The sequencing results revealed that
MsC4H
and
MsCAD
contain 305 and 269 base pairs, which encode 101 and 51
amino acids, respectively. BLAST analysis showed that the cloned nucleotide sequences and their corresponding amino acids are
highly homologous to many
C4H
and
CAD
genes and their encoding proteins from different plant species. The sequences
of
MsC4H
and
MsCAD
had been submitted to Genbank database, whose accession numbers are JQ598686 and JQ598683,
respectively.
Keywords
Miscanthus sinensis
,
C4H
,
CAD
, Cloning, Sequence analysis
Background
At present, 79% of the global energy consumption is
met by fossil energy sources (crude oil, natural gas
and coal) (Soetaert and Vandamme, 2006), which are
the main energy sources to support the global world
economic development and social progress. Due to
limited reserves, it is generally agreed upon that
petroleum will run out within 50 years, natural gas
within 65 years and coal in approximately 200 years at
the present rate of energy consumption. The rapid
increase in fossil energy consumption has resulted in
the environmental and climate problems such as the
greenhouse gas emissions, air pollution and acid rain
(Wuebbles and Jain, 2001). Therefore, countries
around the world are looking for the renewable energy
sources to meet the growing energy crisis and biomass
is one of the most potential renewable energy sources.
An ideal biomass crop is one with a high energy
output whilst requiring minimal inputs (Jones and
Walsh, 2001).
Miscanthus sinensis
belongs to the
genus of
Miscanthus
, subfamily of
Panicoidea
, family
of Poaceae (Zhan et al., 2012), which has a higher
energy yield per hectare than other bioenergy crops
such as willow, poplar, oil and starch crops
(Hastings et al., 2008). Miscanthus has an efficient C4
photosynthesis process, use of water and nitrogen
(Liu et al., 2011). Due to its low mineral content, full
combustion, zero net effect of carbon dioxide,
harmless gas emissions, helpful for the greenhouse
effect alleviation and the soil and water conservation,
resistent to drought, low dependence on water and
fertilizer, easy to harvest (Heaton et al., 2004; Yuan et
al., 2008),
Miscanthus
is destined to become one the
most useful species among the second generation
energy crops. The European Union aims to displace
10% of fossil fuels in the transportation sector with
biofuels in 2020 (Fulton, 2005, http://www.ifri.org/
files/Agriculture/FultontalkIFRI29March.pdf).
Although lignocellulosic biomass is the most