Molecular Cloning and Characterization of Differentially
Expressed Genes in an Aroma Rice Mutant, SA0420
Hsing-Min Su, Chang-Sheng Wang, Ching-Chi Tsai and Wei-Ming Leu
Graduate Institute of Biotechnology
National Chung-Hsing University, Taichung, Taiwan
1
ABSTRACT
SA0420 is a rice mutant generated by imbibing seeds of Tainung No. 67
(TNG67), a popular variety in Taiwan, with the chemical sodium azide
(NaN3) followed with self pollination for ten generations. SA0420
mutant exhibits multiple distinctive traits including a special taro
fragrance in the mature leaves and grains, a dwarf appearance of whole
plant and early maturity. To isolate the differentially expressed genes
related to the above phenotypes, a subtracted cDNA library was
constructed by suppression subtractive hybridization (SSH) using
messenger RNA from mature leaves. Colony arrays containing 9,000
independent clones were screened by hybridizations with
SA0420-specific (tester) and TNG67-specific (driver) probes. Nine
hundred colonies show differential signals larger than three-fold were
re-arrayed and screened by cross-hybridizations to eliminate the
redundant clones. Sequencing and Blast analysis were performed for the
up-regulated candidates. Northern hybridization and RT-PCR are in
progress to verify the significance of these putative clones.
2
INTRODUCTION
Rice is the most important crop for human nutrition in the world (Ning
et al., 2003). It is a staple food for more than one third of the world
population (Wang et al., 2002). Rice is also one of most important crops
in Taiwan. With 430 megabase pair (Mb) in haploid genome, it contains
the smallest genome among the agriculturally important cereals such as
maize, sorghum, barley, and wheat (Ning et al., 2003). Two drafts of
genomic sequences from two cultivated subspecies, Oryza sativa L.ssp.
japonica and Indica, respectively, were already published on 2002 (Goff
et al., 2002; Yu et al., 2002). Therefore, the focus of rice research will be
shifted from structural genomics to the functional genomics. The next
challenge for rice scientists will be to identify and determine functions of
each gene taking advantages of known sequences and resources that open
to public. Owing to the synteny relationship between rice and other
cereals, gene locus revealed from rice research may be applied to
investigation on the Gramineae family (Wang et al., 2002).
Dr. Wang in Taiwan Agricultural Research Institute (TARI) had
adopted mutagenesis by treating seeds with the chemical sodium azide
(NaN3) as the strategy to expand genetic diversity in rice since 1995
(Wang et al., 2002). Tainung No. 67 was chosen for mutagenesis owing to
its superior characteristics and popularity. Among thousands of mutants
that selfed for ten generations after mutation, SA0420 exhibits multiple
distinctive traits including a special taro fragrance in the mature leaves
and grains, a dwarf appearance of whole plant, and early maturity. These
traits are valuable regarding either marketing price (taro fragrance) or
research significance (dwarf, early maturity, rice blast resistance, etc.).
The near-isogenic genetic background of SA0420 with TNG67 provides
an excellent material for comparing their gene expression patterns.
The PCR-based cDNA subtraction method, Suppression Subtractive
Hybridization (SSH, Diatchenko et al., 1996), was used to construct two
differential-expressed cDNA libraries. By arraying ~9,000 colonies on
membranes and re-screening by colony hybridizations, several
up-regulated or down-regulated genes were obtained and under further
investigations. Biochemical functions of the obtained genes were
correlated according not only to their sequence characteristics, but also to
the phenotypes of the segregated F2 progenies. The physiological roles of
genes in plant can be studied right after the gene is obtained without the
3
time-consuming transgenic approach. Furthermore, as the pathway for
production of taro fragrance may have never been turned on in the wild
type rice, novel gene expressions are likely to be detected by this study.
Based on these isogenic rice mutants generated by Dr. Wang, a high
throughput approach for rice functional genomics is established.
4
MATERIALS AND METHODS
Plant materials
Mature leaves of rice are collected from plant grown at field of the
Taiwan Agricultural Research Institute (TARI). TNG67 is a popular
cultivar in Taiwan. SA0420, SA1613, SA1613.1 and SA0428, all contain
taro fragrance to a various extent, are isogenic lines derived from TNG67.
IR5105 is an Oryza sativa L. ssp. Indica.
RNA isolation
Total RNA were isolated using the Total RNA Miniprep Sysytem kit
(Viogene). mRNA were isolated by the PolyATtract mRNA Isolation
System IV (Promega Co.) and reverse transcribed according to
manufacturer’s protocol (Clontech).
Construction of the subtracted cDNA library
Two microliters of driver ds cDNA (600 ng) was added to each of two
tubes containing 2 l of adaptor1- and adaptor 2-ligated tester cDNA(20
ng). The samples were mixed, ethanol precipitated, and then re-suspended
in 1.5 l of hybridization buffer. The solution was overlaid with mineral
oil, the DNAs were denatured (1.5 min, 98C), and then allowed to
anneal for 10 h at 68C. After this first hybridization, the two samples
were combined and a fresh portion of heat-denatured driver (150 ng) in
1.5 l of hybridization buffer was added. The sample was allowed to
hybridize for an additional 10 h at 68C. The final hybridization was then
diluted in 200 l of dilution buffer (20 mM HEPES, pH 8.3/50 mM
NaCl/0.2 mM EDTA), heated at 72C for 7 min and stored at -20C. For
each subtraction, we performed two PCR amplifications. Products from
the secondary PCR reaction were inserted into the pGEM T-Easy
(Promega Co.) using a T/A cloning kit (Invitrogen Co.). The plasmids
were transformed into E. coli Top 10F strain (Invitrogen Co.).
Preparation of colony array
Square plates (22.2 22.2 cm2) containing IPTG and X-gal were dried for
20 minutes right after preparation in laminar flow and were plated out
with colonies (3,000~6,000 colonies/plate) as even as possible. Colonies
formed overnight were picked up by machine (Genetix Q-Pix II) and
arrayed in 384-well culture plates containing medium. After cultured for
5
o/n, bacteria broth was arrayed onto nylon membranes with three spots
per address. Four membranes replicates were prepared simultaneously.
and grew on top of agar for overnight.
Screening of the subtracted cDNA library
Two membranes containing ~9000 colonies were hybridized with
SA0420-specific or TNG67-specific cDNA fragments (digested with
restriction enzymes RsaI, respectively), respectively. The hybridization
signals were recorded by Phosphoimager (BAS 1500, Fujifilm Co.) and
analyzed by Image Gauge program (Fujifilm v. 3.12). Colonies within
10% highest signal differences between two hybridizations were chose
for re-array and second screening.
Southern blotting
Genomic DNA from were extracted from the mature leaves of TNG67
and SA0420, digested by Hind III restriction enzyme, separated on a 1 %
agarose gel, and subjected to hybridization with the 32P-labeled
up-regulated or down-regulated cDNA fragments.
In silico analysis
DNA sequencing and Blast analysis (http://www.ncbi.nlm.nih.gov/BLAST/)
were performed against database containing non-redundant sequences from
various organisms (db-nr), rice genomic DNA (db-wgs, db-htgs, db-gss),
or expressed sequences (db-est) using blastn or blastx programs.
6
RESULTS AND DISCUSSIONS
PCR analysis of the subtraction efficiency
After subtraction, the efficiency for generating TNG67- or SA0420specific cDNA fragments was examined using PCR. The abundance of a
known cDNA (OSGEP1) was compared on samples before and after
subtraction, respectively (Fig.1). Without subtraction, the OSGEP1
trasncript was detectable after amplified by 28 cycles. In contrast, the
house-keeping transcripts were greatly eliminated in the subtracted cDNA
templates and therefore, require 38 cycles of amplification to obtain
product. The upper panel of Fig. 1 showed that the TNG67-specific
cDNA, but not SA0420 samples, were successfully subtracted.
Considering the possibility that the cDNA after subtractions maybe
unequally represented as described (Clontech, PT1117-1), we continued
to check several independently-amplified cDNA populations. By examine
four independently amplified PCR products as templates, the lower panel
of Fig. 1 showed that the SA0420 cDNA fragments were also prepared
successfully.
Examination of the randomness of the subtracted cDNA inserts
PCR-amplified products were inserted into pGEM T-Easy vector
using a T/A cloning kit (Promega Co.) and transformed into E. coli Top
10F strain (InVitrogen Co.). About forty colonies were randomly chose
and their inserts were examined by Eco RI digestions. As shown by Fig. 2,
the size of inserts range between 0.1~0.8 kb and the major population are
between 0.3~0.6 kb. Furthermore, the blue colonies, but not the light blue
ones, did not contain inserts (data not shown).
Screening of the differentially expressed clones by colony
hybridizations
Using the colony picker (Genetix-Q Pix II), ~9,000 colonies were
arrayed on nylon membranes. Two sets of blot, with each containing 24
membranes, were hybridized with 32P-labeled SA0420-specific or
TNG67-specific cDNA probes, respectively. Fig. 3 shows one set of the
hybridization result. Some colonies with significant signal differences can
be recognized even by eyes. In order to obtain a systematic data, the
hybridization signals were recorded by PhosphoImager and quantified by
Image Gauge program. The signal ratios between hybridizations with
7
SA0420 or TNG67 probes were calculated by Excel program (Fig. 4). We
chose colonies exhibit signal differences within the highest 10% for
further analysis. About 890 colonies were re-arrayed on three membranes
in 384-well format. With those re-arrayed membranes, similar
hybridization experiments were performed again and the clones were
ordered according to their signal differences. Clones with higher signal
difference have higher priority for sequencing and analysis.
Elimination of the redundant clones
Firstly, DNA sequencing and Blast searches were applied to several
randomly chose colonies. Using the sequenced fragments as probes,
hybridizations were performed with the re-arrayed membranes to
eliminate redundant clones corresponding to the same genes. This
procedure was repeated several times by mixed probes to eliminate the
redundant clones as complete as possible. In total, about 357 clones
shows hybridization signals to probes composed of 46 independent gene
fragments during 10 hybridizations. Among the 895 colonies, one probe
shows the highest redundancy and was chose for further analysis as
described below.
Selection of the most promising candidate
One cDNA sharing high sequence homology to a known receptor
kinase, Xa21, cross-hybridized with other 97 colonies and account for
~10% of our sample under analysis (Fig. 5). We designated this gene as
Xa21-1. All those 97 colonies were found only in samples originated from
SA0420-specific (the upper two 384-well blots) but not TNG67-sepcific
populations. This observation suggests that the subtracted cDNA library
was successfully constructed and the data was accurately analyzed.
Xa21 is a membrane-bound receptor-like kinase (RLK) with
serine/threonine specificity and can confer resistance to the bacteria leaf
blight in rice (Song et al., 1995). Xa21-mediated resistance conforms a
gene-for-gene interaction in which Xa21 expressing plants are resistant to
Xanthomonas oryzae pv. Oryzae race 6 strain coding for a corresponding,
yet uncharacterized, Avr product. When expressed in rice cell cultures,
Xa21 retained its recognition specificity and its functionality in inducing
defense responses such as H2O2 production, changes in gene expression,
and induction of cell death after incubation with the incompatible X.
oryzae pv. oryzae strain (Romeis, 2001).
8
Besides the SA0420-specific cDNA fragments, putative clones that is
TGN67-specific were also obtained by similar procedure. Interestingly,
another Xa21-like sequence, sharing 82% sequence identity with Xa21-1,
was obtained and designated as Xa21-2. Coincidently, the clones
cross-hybridized with Xa21-2 were appeared only in the TNG67-specific
populations, without overlapping with the one hybridized with Xa21-1
probe. This result shows that both probes are gene-specific in colony
hybridization.
Xa21-1 is up-regulated but Xa21-2 is down-regulated in SA0420
In order to confirm the differential expression of Xa21-1 and Xa21-2
in SA0420 and TNG67, we performed RT-PCR analysis on six lines of
rice (except for IR5105, SA0420, SA0418, SA1613 and SA1613.1 are all
aromatic mutants derived from TNG67 independently). PCR using
Xa21-1- or Xa21-2-specific primers were performed for 25 or 30 cycles
and the amplified products were detected by hybridization. As shown by
Fig. 6, the transcripts of actin gene are more or less equal among different
samples except a little bit less in SA0418. However, Xa21-1 transcripts
were only detectable in SA0420 but absent in the other lines. After 30
cycles of amplification, only slight amount of Xa21-1 could be detected
in the SA0418, SA1613, SA1613.1 samples, but still no signals at all in
the TNG67 or IR5105 lines. In contrast, Xa21-2 exhibit an opposite
expression pattern that only the wild type TNG67 express Xa21-2 to a
substantial amount. We conclude that Xa21-1 is up-regulated but Xa21-2
is down-regulated in SA0420 rice mutant.
Selection of more candidates
About 26 genes shows high differences in expression signals were
sequenced and blast analyzed against GanBank (Table. 1). No similar
biochemical roles or physiological phenomenon can be concluded from
their sequence information.
Perspective
Future experiments including RT-PCR, Northern hybridization,
genomic Southern, transgenic studies and F2 progeny analysis, etc. will
be performed to elucidate functions of the differentially expressed genes
and to correlate their roles with the phenotypesof SA0420.
9
Fig. 1
Fig. 2
Fig. 3
10
Fig. 4
Fig. 5
11
Fig. 6
Table 1
Clone
Blast search
Chromosome location
1-40
glycine-rich cell wall structural protein 2 precursor
# 10
2-13
root border cell-specific protein
No genomic clone, maybe a fused clone
2-15
ankyrin-like protein
#1
2-16
unknown protein
# 10
2-19
no significant similarity found
#4
2-20
putative diterpene cyclase
unknown
2-25
beta-galactosidase
unknown
2-26
putative protein kinase Xa21 (Xa21-1)
unknown
2-27
artificial clone
-
2-28
hypothetical protein
#3
2-29
L-ascorbate peroxidase
#3 (?)
2-30
a strange clone
unknown
2-34
phosphoethanolamine methyltransferase
unknown
2-38
OsD305 at 5’but maybe a fused clone
# 4 and # 12
2-40
5 is similar to wak1 gene
5 from # 1 and 3 from #6
but 3 is cell wall-associated kinase
2-42
putative ribonucleoprotein
#7
2-44
pullulanase
#4
2-45
senescence-associated protein-like protein
unknown
2-50
50% similar to KH domain protein
unknown
2-51
P0436E04.7 (an unknown protein with EST)
#1
5-13
low similar with a rice putative protein
unknown
12
9-1
hypersensitive-induced reaction protein
unknown
9-6
putative protein kinase Xa21 (Xa21-2)
unknown
9-54
short region similar to a rice putative protein
unknown
9-80
vector sequence
-
9-85
putative protein
unknown
13
Fig. 1. PCR analysis of the subtraction efficiency. OSGEP1-specific primers (E9
and E22) were PCR against templates of SA0420-specific or TNG67-specific cDNA
fragments for 23, 28, 33, or 38 cycles. The products were resolved on 2% agarose gel
and hybridized with OSGEP1 probe. The size of OSGEP1 fragment is 738 bp.
Fig. 2. Check the randomness of the sub-cloned fragments. Colonies containing
the subtracted cDNA fragments were randomly chose for plasmid isolation. The
plasmid were digested by EcoRI and resolved by 2% agarose gel with the molecular
weight marker loaded at the left.
Fig. 3. Screening of the differentially expressed clones by colony hybridizations.
Two blots harboring 384 addresses with triplicate colonies in each address were
prepared simultaneously and hybridized with 32P-labeled SA0420-specific or
TNG67-specific cDNA probes, respectively. Colonies showing distinct signal
differences between two hybridizations were marked by circle.
Fig. 4. Quantitative analysis of the hybridization signals. Hybridization signals of
~9,000 independent clones arrayed on 24 membranes were recorded by
PhosphoImager and quantified by Image Gauge program. The signal ratios between
hybridizations with SA0420- or TNG67-specific probes were calculated by Excel
Program. Type A were marked for clones that shows SA0420>>TNG 67 and with
high original signals (may represent the abundant transcript that is up-regulated in
SA0420); type B: SA0420>>TNG 67 but low in the original signal (may represent the
rare transcript that is up-regulated in SA0420); type C: SA0420<<TNG 67 (may
represent the down-regulated transcript in SA0420).
Fig. 5. Elimination of the redundant clones –example of Xa21-1 and Xa21-2. Two
set of re-arrayed membranes containing 890 independent colonies were hybridized
with Xa21-1 (left panel) or Xa21-2(right panel) probes isolated in this study. Colonies
that shows cross-hybridization signals are eliminated from list for further analysis.
Fig. 6. RT-PCR examination of the differentially expressed Xa21-1 and Xa21-2.
RNA from mature leaves of the TNG67, SA0420, SA0418, SA1613, SA1613.1, or
IR5105, respectively, were reversed transcribed. Using Xa21-1 or Xa21-2 specific
primers, PCR were performed against the above templates for 25 or 30 cycles.
Primers for actin genes were used for internal control. The amplified bands were
visualized by hybridization with their corresponding probes.
14
Table 1. Blast analysis of the candidate genes.
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