全 文 ：作物学报 ACTA AGRONOMICA SINICA 2009, 35(11): 2022−2028 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This study was financially supported by the Fujian Natural Science Foundation (2009J05050), the National High Technology Research and Develop-
ment Program of China (863 Program) (2007AA100701), the Program of Introducing International Super Agricultural Science and Technology (948
Program) (2006-G37), National Science and Technology Project File of Science and Technology Commission, Fujian Science Department
(F2007AA100701), the earmarked fund for Modern Agro-industry Technology Research System.
*
通讯作者(Corresponding authors): XU Li-Ping and ZHANG Mu-Qing.
第一作者联系方式: E-mail: queyouxiong@yahoo.com.cn
Received(收稿日期): 2009-02-18; Accepted(接受日期): 2009-06-25.
DOI: 10.3724/SP.J.1006.2009.02022
Isolation and Characterization of Disease Resistance Gene Analogs from
Erianthus arundinaceus cDNA
QUE You-Xiong, XU Li-Ping*, LIN Jian-Wei, XU Jing-Sheng, ZHANG Mu-Qing*, and CHEN Ru-Kai
Key Laboratory of Sugarcane Genetic Improvement, Ministry of Agriculture / Fujian Agriculture and Forestry University, Fuzhou 350002, China
Abstract: Plant disease resistance genes (R-genes) encode some conserved motifs. According to the conserved motifs present in
the known NBS-LRR R-gene sequences and R gene analogs (RGAs), several degenerate primers were designed and applied in the
RGA isolation from Erianthus arundinaceus using PCR approach. In total, 6 RGAs were successfully obtained, with GenBank
accession numbers of EU685835, EU685836, EU685837, EU685838, EU685839, and EU685840. Multiple alignments showed
that the encoding sequences of the six clones were highly conserved and strikingly similar to the eleven most typical NBS-LRR
type R-gene peptide sequences, especially at the four NBS motifs of P-loop, kinase-2, kinase-3a, and HD. The identity percentage
at the amino-acid level ranged from 8.3% to 93.0% among all 17 sequences tested and from 30.5% to 45.6% among the six RGAs
cloned in this study. The results of cluster analysis and the existence of an aspartic acid residue (D) at the final residue position of
the kinase-2 motif also indicated that all of E. arundinaceus RGAs might belong to non-TIR group. The Real-time PCR results
showed that all of the RGAs were constitutively expressed in roots, stalks and leaves of E. arundinaceus, and their expression
could be up-regulated in leaves by the exogenous signal molecules SA and H2O2. Therefore, it suggested that E. arundinaceus
RGAs might play important roles in disease resistance in an SA- and H2O2-dependent defense response pathway. Further studies
should aim to clone full-length R-genes in E. arundinaceus and characterize their functions in defense responses.
Keywords: Erianthus arundinaceus; Resistance gene analogs (RGAs); Degenerate primers; Real-time PCR
斑茅 cDNA 中抗病基因同源序列的分离和表达特性分析
阙友雄 许莉萍* 林剑伟 徐景升 张木清* 陈如凯
福建农林大学 / 农业部甘蔗遗传改良重点开放实验室, 福建福州 350002
摘 要: 植物抗病基因具有一些特定的保守结构域。本研究根据已知植物同源抗病基因(RGAs)保守序列设计简并引
物, 从甘蔗近缘植物斑茅的 cDNA 中扩增出 6 条抗病基因同源序列, 它们在 NCBI 上登录号分别为 EU685835、
EU685836、EU685837、EU685838、EU685839 和 EU685840。序列分析表明, 这些 RGAs 均含有典型的 NBS-LRR
类抗病基因保守结构域 P-loop、kinase-2a、kinase-3a和疏水结构域(hydrophobic domain, HD)。氨基酸序列的同源性
比对表明，6 条 RGAs 序列同 11 条参试的抗病基因之间的同源性为 8.3%~93.0%，而 6 条 RGAs 之间的氨基酸序列
同源为 30.5%~45.6%。另外，本实验所克隆的 6条斑茅抗病基因同源序列中, kinase-2 (LLVLDDVW/D)最后一个氨基
酸皆为色氨酸，推测所克隆的 NBS-LRR 类抗病基因都属于 non-TIR-NBS-LRR类。定量 PCR分析表明, 6条斑茅抗
病基因同源序列在根、茎和叶片中组成型表达，同时这些抗病基因同源序列的表达会受外源信号分子水杨酸和过氧
化氢的上调作用，可能在斑茅的抗病性中具有一定的作用。
关键词: 斑茅；抗病基因同源序列；简并引物；定量 PCR
Plants have developed complex defense mechanism
during evolution to resist the various harmful pathogens
they encountered. The mechanism involves interaction of
the plant R-gene products with the various components
from the pathogens. Therefore, the isolation and charac-
terization of disease R-genes has great significance for
understanding the complex mechanisms of resistance, the
interactions involved in pathogen recognition, and also
第 11期 阙友雄等: 斑茅 cDNA中抗病基因同源序列的分离和表达特性分析 2023


the evolution of the R-genes. Furthermore, the isolated
R-genes can be transferred to target plant species to im-
prove corresponding resistance [1].
Till now, a number of disease R-genes from various
plant species have been cloned. Based on their conserved
domains, R-genes can be classified into four classes[2-4].
Among them, NBS class R-genes accounting for almost
71% of the cloned R-genes, including RPS2[5], RPM1[6],
RPP5[7], N[8], Mi[9], L6[10] and so on. NBS domain can
bind ATP or GTP and plays important roles in signal
transduction of plant resistance response. Degenerate
primers designed according to the conserved NBS domain
have allowed successful PCR-amplification of multiple
DNA sequences from a number of plant species that share
striking similarity to the NBS-LRR (leucine-rich repeat)
class of R-genes[11-15]. These sequences have been called
resistance-gene analogs (RGAs)[11] or resistance-gene
candidate (RGC) sequences[14]. Genetic analysis has asso-
ciated a number of these sequences to known gene loci
that confer resistance to viruses, bacteria, fungi, or nema-
todes[12-16]. Interestingly, some of these sequences appear
to be the true resistance genes.
Erianthus arundinaceus is one of the most important
wild relatives of sugarcane, which has great potential as a
contributor of germplasm to current sugarcane breeding
for better ratoon ability and vigor, tolerance to environ-
mental stresses such as drought and flooding, and disease
resistances including root rot caused by Pachymetra
chaunorhiza Croft and Dick [17-18] and so on. To introduce
these desirable characters into sugarcane cultivars, nu-
merous attempts have been made by crossing E. arun-
dinaceus and sugarcane. However, when the E. arun-
dinaceus was used to broaden the genetic base of modern
sugarcane and to introduce desirable traits, some un-
wanted traits were also brought into the target sugarcane
variety, thus limiting the success of these introgression
programs. Two approaches were proposed to overcome
this problem. One is elimination of these undesirable
traits through multi-generation backcross at the expense
of time, and labor. However, most of the progeny of cross
between E. arundinaceus and sugarcane are male sterile.
The other is to clone the useful genes controlling the tar-
get traits and transferred them to target sugarcane varie-
ties for trait improvement. However, there is no reported
effort on the cloning of RGAs from E. arundinaceus
cDNA.
In this study, degenerate primers were designed ac-
cording to the sequences of the P-loop and the hydropho-
bic domain (HD) in the conserved NBS domain of some
typical R-genes. Then, PCR-based approach was applied
to clone NBS-LRR class RGAs from E. arundinaceus.
Finally, sequence analysis and the expression pattern of
these RGAs were carried out by bioinformatics tool and
Real-time PCR method, respectively. These studies
should provide valuable information for the ultimate
cloning of E. arundinaceus resistance genes.
1 Materials and Methods
1.1 Plant materials and RNA extraction
Erianthus arundinaceus, a wild relative of Saccharum
officinarum, was used for RGAs cloning and the corre-
sponding expression analysis. Leaves of seedlings were
used in the following treatments. Some leaves were
sprayed with 2.5 mmol L−1 salicylic acid (SA) and 10
mmol L−1 H2O2, respectively. Control leaves were
sprayed with distilled water. After 72 h treatments, the
roots, stalks, and leaves were collected and used for RNA
extraction. Total RNA was extracted with improved
TRIZOL method[19]. Subsequently, total RNA was used
to produce cDNA using M-MLV reverse transcriptase
(TaKaRa) according to the manufacturer’s instruction.
RNA from leaves was especially useful for RGAs clon-
ing due to the SA-induced up-regulation expression of
RGAs[20].
1.2 Primers design and PCR analysis
Based on the conserved motifs in the aligned amino
acid sequences of the known NBS-LRR R-genes and
RGAs, seven degenerate primers were designed corre-
sponding to the P-loop motif and HD motif[11,21]. The
primers sequences were shown in Table 1. PCR amplifi-
cation was carried out in an Eppendorf (model info)
thermocycler with the same protocol and programme as
reported before [22]. Amplified cDNA fragments were
separated on a 1.5% agarose gel, and recovered with a
DNA Gel Extraction Kit (Sangon). The purified cDNA
fragments were cloned and the corresponding positive
clones were sequenced.

Table 1 Primers used in RGA amplification
Primer
code
Conserved
motif Primer sequence (5–3)
a
H1145 GGVGKTT GGIGGIRTIGGIAAIACIAC
H2017 GGSGKTT GGIGGIWSIGGIAARACIAC
H2018 GGLGKTT GGIGGIYTIGGIAARACIAC
H2019 GGMGKTT GGIGGIATIGGIAAAACIAC
H2021 GLPLAL CAACGCTAGTGGCAATCC
H2027 GLPLAL YTCIGGRAAIARIGCRCARA
LM637 GLPLAL ARIGCTARIGGIARICC
a I: inosine; R: A/G; W: A/C; Y: C/T; N: A/G/C/T; S: G/C; D:
A/G/T; K: G/T.

1.3 Sequence and phylogeny analysis
Sequence assembly and analysis were conducted with
DNAstar program, and tBLASTx search of the protein
database of GenBank was performed for the assembled
sequences. BLASTn search in GenBank was performed
if tBLASTx search in the protein database failed to re-
veal homology with cloned R-genes or RGAs. The ORF
Finder program in NCBI was used to search ORF of E.
arundinaceus RGAs. Multiple sequence alignments of
amino acid sequences deduced from E. arundinaceus
2024 作 物 学 报 第 35卷

RGAs with cloned typical NBS-LRR type R-genes were
carried out with MegAlign program of DNAstar with
default options, and phylogenetic tree was drawn ac-
cordingly[22,23]. The eleven R-genes used in multiple
alignment were the following: RPS2(ATU1415), RPS5
(AF074916), RPP8(AAC83165), Xa-1(AB002266), Pib-
(AB013448), N(U15606), L6(LUU27081), RPM1(AF1-
22982), I2C(AF004878), Mi(AF091048), and Dm3(AF-
072267).
1.4 Expression analysis by Real-time PCR
cDNA synthesis was carried out using ExScript
RT-PCR Kit (TaKaRa) according to the protocol pro-
vided. The reverse transcription reaction protocol and
programme were as described by Que et al.[22]
Specific primers for 25S rRNA and E. arundinaceus
RGAs were designed accordingly. Then, Real-time PCR
analysis of them in cDNA samples were conducted in a
Mini Opticon Real Time PCR System (Bio-Rad), with
the same protocol and programme as described in Que et
al.[22] Data was analyzed through DPS software. The se-
quences of primers used in Real-time PCR detection
were shown in Table 2.

Table 2 Primers used in Real-time PCR detection
Gene code (Accession numbers) Forward primer (5–3) Reverse primer (5–3)
cRGA-Q1 (EU685835) GGAGTTGCTGTGTCTGCCTA TTGCACTAGCTTCGCAACTT
cRGA-Q2 (EU685836) GGAGTTGCTGTGTCTGCCTA TTGCACTAGCTTCGCAACTT
cRGA-Q3 (EU685837) CAGAGCAGAGAGCGAGTTTG ACCAAGCATCTTTCTCCCAT
cRGA-Q4 (EU685838) AAAGGGAACTTCAGCAAACAA TCCAACATCTTCATCTTGCAC
cRGA-Q5 (EU685839) ATTGGAATGGACATTGTTCG TTCAGCAGGAAGATTGGTCA
cRGA-Q6 (EU685840) ACTGCCAGCAACAGAATACG TCATTATTTGCCAGCTCACC
25S rRNA(BQ536525) GCAGCCAAGCGTTCATAGC CCTATTGGTGGGTGAACAATCC

2 Results
2.1 Cloning of NBS-LRR type RGA from E. arun-
dinaceus
As shown in Fig. 1, several RGAs were amplified
from E. arundinaceus cDNA. While some primer pairs
resulted in amplified products with several bands of dif-
ferent lengths, most primer pairs produced a single band
of the expected size deduced from the cloned NBS-LRR
type R-genes. PCR products with the expected size were
obtained from some pairs of degenerate primers. Only
the primer combination of H2016/H2021 yields a distinct
strong band with the length of 500–750 bp (data not
shown), and the lengths of most amplification products
from tested primers were about 500 bp. Then, PCR
products of the expected size from the primer pairs, plus
the band with length of 680 bp, were cloned for se-
quencing. These fragments were named as cRGA-Q1,
cRGA-Q2, cRGA-Q3, cRGA-Q4, cRGA-Q5, and cRGA-



Fig. 1 RGA amplificataion from E. arundinaceus
Primers H1145-H2010 for the first lane; H2017-H2021 for the
second lane; H2018-H2027 for the third lane; H2018-H2027 for the
fourth lane and fifth (negative control).
Q6, of which the actual length based on the se quences
was 513, 672, 668, 516, 516, 516, 528, and 534 bp,
respectively.
2.2 Sequence and phylogenetic analysis of RGA
from E. arundinaceus
One of clones from each of the feasible primer com-
binations was sequenced. In total, six RGAs were suc-
cessfully obtained, with GenBank accession numbers of
EU685835, EU685836, EU685837, EU685838,
EU685839, and EU685840. All of the six RGAs could be
translated into polypeptides without any stop codon.
Therefore, these clones were included in further analysis.
Searches of GenBank database using BLASTx algorithm
revealed that they showed strong overall similarities to
several types of plant R-gene sequences and many RGA
or RGC sequences cloned from other plant species using
similar PCR-based approaches (BLAST search data not
shown). Multiple alignments performed with these six
E .arundinaceus RGAs showed that they were highly
conserved and strikingly similar to the eleven most typi-
cal NBS-LRR type R-gene peptide sequences, especially
at the four NBS motifs of P-loop, kinase-2, kinase-3a and
HD (Fig. 2). As showed in Fig. 2, the highest homology
among all E. arundinaceus entries is with the four NBS
motifs: the P-loop (GMGGVGGKTT), the kinase-2
(LIVLDD), the kinase3a (GSR/KILVIIR) and the HD
(GLPLAL). These conserved NBS motifs are featured in
typical NBS-LRR type R-genes, such as N, RPS2’ and L6.
While the DNA sequences corresponding to the 5-P-loop
and a portion of the 3-HD (hydrophobic domain)
第 11期 阙友雄等: 斑茅 cDNA中抗病基因同源序列的分离和表达特性分析 2025




Fig. 2 Alignment of the deduced amino-acid sequences of RGAs from E. arundinaceus with the NBS domains of
eleven known R-gene
2026 作 物 学 报 第 35卷

region in the PCR product were derived from primers
and may not be the exact sequences in E. arundinaceus
genome. The two internal motifs, kinase-2 (LIVLDD)
and kinase3a (GSR/KILVIIR), are independent of the
primer sequences used in the PCR reaction. Therefore,
the six sequences belong to the NBS-LRR R-gene su-
per-family. It has also been proposed that a single residue
in the highly conserved motif (LLVLDDVW/D) known
as kinase-2 within the NBS can be used to predict the
presence or absence of the TIR domain with 95% accu-
racy, i.e. a tryptophan (W) residue in non-TIR-NBS-LRR
type whereas an aspartic (D) residue in TIR-NBS-LRR
type. Our sequence results showed that all of the cloned
RGAs contained the residue W in LLVLDDVW/D motif.
From above, it is likely that all the RGAs cloned in this
study belong to the non-TIR-NBS-LRR type.
Using the MegAlign program in DNAStar, pair-wise
comparisons of the translatable sequences of six RGAs
cloned in this study with the corresponding amino acid
regions of eleven cloned NBS-LRR type resistance genes,
was conducted. As shown in Fig. 3, while the identity
percentage at the amino-acid level ranged from 8.3% to
93.0% among all 17 sequences tested, the identity per-
centage among six RGAs cloned in this study ranged
from 30.5% to 45.6%. Phylogenetic analysis was further
performed to evaluate the relationship among E. arun-
dinaceus RGAs and cloned NBS-LRR type R-genes. As
can be seen from Fig. 4, cRGA-Q1, cRGA-Q2, cRGA-
Q4, cRGA-Q5, together with I2C, RPS8, Mi, and Xa1
formed one major cluster. The other major cluster con-
tained cRGA-Q1, cRGA-Q6, N, RPP5, L6, and RPM1.
The remaining three sequences formed another two clus-
ters, in which RPS2 and RPS5 formed one cluster and
Dm3 alone formed the other cluster, respectively.



Fig. 3 Similarity comparison of E. arundinaceus RGAs with NBS domains of eleven known R-genes



Fig. 4 Phylogenetic tree based on alignment of the deduced
amino-acid sequences of six RGAs and NBS domains of eleven
known R-genes


2.3 Expression of E. arundinaceus RGAs under
SA and H2O2 treatments
Real-time PCR method was used to examine the ex-
pression pattern of six RGAs from E. arundinaceus in
response to SA and H2O2 treatments, respectively. The
results presented in Fig. 5 indicated that the mRNA ac-
cumulation of all the six RGAs cloned from E. arun-
dinaceus increased both upon SA and H2O2 treatments,
especially for the expression of cRGA-Q1, cRGA-Q3,
and cRGA-Q6 under SA treatment and that of cRGA-Q1
and cRGA-Q5 under H2O2 treatment. According to the
above analysis, E. arundinaceus RGAs, up-regulated in
response to SA and H2O2 treatments, may function during
the pathogen attack.
2.4 Expression of RGAs in various tissues of E.
arundinaceus
Real-time PCR was also performed to investigate the
tissue-specific expression of all these RGAs from E.
arundinaceus. Interestingly, all of six RGAs cloned here
were found to be constitutively expressed in E. arun-
dinaceus, high in leaf, moderately in stalk and low in root.
The expression of RGA-Q1 was depicted in Fig. 6 for
example. This pattern of expression was consistent with
the fact that most disease resistance genes were constitu-
第 11期 阙友雄等: 斑茅 cDNA中抗病基因同源序列的分离和表达特性分析 2027




Fig. 5 Expression of the RGAs from E. arundinaceus in response to SA and H2O2 treatments

tively expressed to prepare for pathogen attack. This
suggests that the six RGAs cloned here may relate to
disease resistance in this aspect.



Fig. 6 Expression of cRGA-Q1 in different tissues of
E. arundinaceus

3 Discussion
Due to the lack of adequate genetic and molecular re-
sources, it is still difficult to isolate a resistance gene
through positional cloning or transposon tagging from E.
arundinaceus. Isolation, characterization and expression
analysis of RGAs in E. arundinaceus should give us
clues to clone E. arundinaceus resistance genes. Up to
the present, there is not any report on the isolation and
characterization of RGAs in E. arundinaceus cDNA. In
this study, we have successfully cloned six RGAs from E.
arundinaceus cDNA. It should be pointed out that, when
using genomic DNA as template in PCR amplification,
the corresponding RGAs may contain introns, and all
RGAs cloned from cDNA should be coding sequences
without any stop codons [22]. All of six RGAs cloned here
showed high similarity to eleven typical NBS-LRR type
R-gene peptide sequences, especially at the four NBS
motifs of P-loop, kinase-2, kinase-3a and HD. They do
not contain any intron and were coding regions. On the
other hand, nucleotide binding site (NBS) is just a con-
served domain of plant R-genes. Not only has it been
found in disease resistance genes, but this domain also
have been found in ATP and GTP binding proteins [22].
Besides NBS, E. arundinaceus NBS-LRR type R-genes
should have other important domains, such as Toll or
Interluken-1-receptor (TIR) or Leucine-rich repeats
(LRR). Therefore, the six NBS fragments were only po-
tential resistance gene analogs in E. arundinaceus.
Several researchers performed extensive analysis of
the conserved domains of cloned plant NBS-LRR classR-
genes, and suggested that NBS-LRR type R-genes could
be divided into two groups: TIR-NBS-LRR group and
non-TIR-NBS-LRR group[14,24]. And they also found that
monocot lacks a family of NBS-LRR genes with a TIR
motif [21]. Further, the final residue position of the
kinase-2 motif (LLVLDDVW/D) could be used in
TIR-NBS-LRR group and non-TIR-NBS-LRR division,
with an accuracy of 95%, an aspartic acid residue (D) in
the TIR group and a tryptophan residue (W) in the
non-TIR group [21]. Our data indicated that an aspartic
acid residue (D) at the final residue position of the
kinase-2 motif was present in all the six RGAs isolated
from E. arundinaceus. It suggests that all of the E. arun-
dinaceus RGAs might belong to non-TIR group. The full
length cDNA sequence information of six RGAs is
needed for the classification of these E. arundinaceus
RGAs regarding TIR or non-TIR group. TIR domains are
thought to have function in signal transduction or patho-
gen recognition [25]. The absence of TIR domains may
suggest the loss of TIR-related defensive signal pathways
in E. arundinaceus.
SA and H2O2, the two most important mechanical fac-
tors, influence a series of gene expression during the
pathogen attack and are involved in the expression of
localized hypersensitive reaction (HR) as well as sys-
temic acquired resistance (SAR) [26-27]. They can not only
function as a critical signal for downstream R-genes but
also up-regulate the expression of certain R-genes [27-28].
Application of exogenous SA has long been known to
activate expression of pathogen-related (PR) genes and to
induce resistance to plant diseases[20,29]. It was reported
that H2O2, as a signal factor, functioned in the resistance
elevation through its activation of some defense mecha-
nism at the early stage of intimidation[27]. In addition,
most of the cloned plant disease resistance genes are ex-
pressed constitutively and at a very low level[30]. In our
study, all six RGAs were constitutively expressed in roots,
stalks and leaves of E. arundinaceus, and their expres-
sion could be up-regulated in leaves by the exogenous
signal molecules, SA and H2O2. The above results sug-
gested that E. arundinaceus RGAs may play certain roles
during disease resistance in an SA- and H2O2-dependent
defense response pathway. The ultimate cloning of resis-
tance gene should shed light on the utilization of E.
arundinaceus in the resistance improvement in sugarcane
breeding program. Hence, further research should aim to
2028 作 物 学 报 第 35卷

clone full-length R-genes in E. arundinaceus and study
their functions in defense responses.
4 Conclusions
Six RGAs cloned belong to non-TIR type RGAs and
their expression is constitutive in roots, stalks, and leaves
of E. arundinaceus, while their expression can be
up-regulated by the exogenous signal molecules, SA and
H2O2. The RGAs cloned here may play certain roles in E.
arundinaceus disease resistance.

Acknowledgments: Thanks to Que Qiudeng in Syn-
genta for his kind help during the manuscript preparation.
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