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Molecular Plant Breeding 2011, Vol.2, No.9, 60
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67
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65
expression in wheat and sorghum as in rice and maize
(Dian et al., 2003; Jiang et al., 2004; Yan et al., 2009).
In addition, those SS duplication and expression
divergence were similar to the starch legumes (mung
bean and cowpea) genomes (Pan et al., 2009). In
summary, the present study and previous reports
indicate that duplicators of
GBSS
Ⅰ
,
SS
Ⅱ
, and
SS
Ⅲ
,
but not
SS
Ⅳ
, were remained in the genome and
diverged in expression in all observed Gramineae
species.
2 Materials and methods
2.1 Plant materials and growth conditions
The sorghum (
Sorghum bicolor
L.) and common
wheat (
Triticum aestivum
L.) variety Shi4185 were
planted in trail field in South China Botanical Garden,
Chinese Academy of Sciences, Guangzhou, and P. R.
China. The seeds were harvested four times after
flowering, while the roots were got from the fifth day
after germinate (DAG) seeding and full expanded
leaves were taken from seedling at one day after
flowering. Samples were frozen under liquid nitrogen
and stored at
-
72
℃
until use. All samples were
collected in a time period between 9:00 am to 10:00 am.
2.2 cDNA cloning of
GBSS
,
SS
Ⅱ
, and
SS
Ⅲ
genes
in sorghum and wheat
Total RNA was isolated from sampled leaves with
Plant RNAout kit (Tiandz Company, http://www.tian-
dz.com). First-strand cDNA synthesis using M-MLV
reverse transcriptase (Promega, http://www.promega.
com) following manufacture's protocol. A specific
fragment of sorghum and wheat
GBSS
,
SS
Ⅱ
, and
SS
Ⅲ
genes were amplified with the primer pair (Table 2),
designed based on the conserved regions of the
corresponding genes from other higher plants. The
purified fragments were cloned into a pMD18
-
T
vector (TaKaRa, http://www.takara.com.cn) and
confirmed by sequencing in the Invitrogen Company
(http://www.invitrogen.com.cn). The 5'
-
and 3'
-
ends
of sorghum
GBSS
,
SS
Ⅱ
, and
SS
Ⅲ
genes were
obtained with a 5' full RACE cDNA Amplification kit
(Clontech) according to the user's instructions. Based
on these sequences, 5’
-
end of the
TaSS
Ⅱ
b
and
TaSS
Ⅲ
b
cDNAs was determined by using the BD SMART™
RACE cDNA Amplification Kit (Invitrogen, Carlsbad,
CA, USA). Putative transit peptide were identified
using the ChloroP neural network analysis of the 100
amino acids at the N terminus of each sequence
(http:// www.cbs.dtu.dk/ services/ChloroP).
2.3 Semi-quantitative RT-PCR analysis
Total RNA was extracted from roots, leaves and
developing endosperms using the Plant RNAout kit.
PCR amplifications were performed on the first cDNA
strand using their specific primer sets (Table 2; Table 3).
Primers (Table 2; Table 3) that amplify sorghum Actin
(X79378) and wheat Actin (AY663392) were used as
a control. PCR products were analyzed by 1% agarose
gels, with ethidium bromide staining and take photo
through Fluorescence Chemiluminescence & Visible
Imaging System. Afterwards the purified fragments
were cloned into the pMD18
-
T vector and the
sequence were determined.
2.4 Data analysis
Phylogenetic analysis was carried out using the
conserved domains sequences of
SS
genes.
Alignments of the SS sequences were aligned using
the ClustalW program on EBI Web server. Phylo-
genetic trees were calculated using PhyML Online
analysis (Guindon et al., 2005) (http://mobyle.
pasteur.fr/cgi-bin/MobylePortal/portal.py?form=phyml)
based on the JTT model with a constant rate of site
variation (Guindon and Gascuel, 2003). Bootstrap
values were calculated from 100 replicate analyses.
2.5 Gene loci comparison
To confirm their physical location, the gene loci on
rice and sorghum were obtained by the alignment of
the cDNA sequences from the NCBI database (http://
www.ncbi.nlm.nih.gov/blast) to the corresponding
chromosome-based pseudomolecules using PHYTO-
ZOME blast (http://www.phytozome.net/search.php?
show=blast). The location of the maize genes on the
chromosome pseudomolecules was traced though the
anchored corresponding BAC genome sequences or
related markers (http://www. maizegdb.org). Finally,
the loci of anchored rice, maize and sorghum starch
synthesis genes were compared on the rice/maize/
sorghum synteny physical maps available at Gramene
(http://www.gramene.org/).