全 文 ：Received 17 Apr. 2003 Accepted 14 Sept. 2003
Supported by the National Natural Science Foundation of China (30100014).
* Author for correspondence. Tel: +86 (0)10 62893462; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 497-504
Non-coding RNA for ZM401, a Pollen-specific Gene of Zea mays
DAI Xiao-Yan, YU Jing-Juan*, ZHAO Qian, ZHU Deng-Yun, AO Guang-Ming
(State Key Laboratory for Agricultural Biotechnology, College of Biology, China Agricultural University, Beijing 100094, China)
Abstract : In our previous study, a cDNA library to poly(A) RNA isolated from mature pollen of Zea mays
L. was constructed. One cDNA fragment, designated ZM401 (Z. mays), was obtained from maize mature
pollen cDNA library by differential screening and cold-plaque screening method. It was specifically or
preferentially expressed in mature pollen. According to the ZM401 cDNA fragment sequence, full length of
ZM401 cDNA was generated by 5 and 3 RACE methods. ZM401 cDNA was 1 149 bp in length. Within the
full-length cDNA sequence, a clear open reading frame (ORF) was undectable by OMEGA 2.0 and DNAMAN
softwares. The longest potential ORF was 269 nucleotides (791–1 060), coding 89 AA, had a poor consensus
sequence for translation initiation. But it had a poly(A) tail. All these results suggested that ZM401 was one
of a growing number of non-coding mRNA-like RNA transcripts that exerted their cellular functions directly
as RNA. RT-PCR and Northern blotting analyses showed the ZM401 was transcribed from tetrad stage of
microspore development and increased in concentration up to mature pollen, which suggested that ZM401
belongs to late gene in pollen development. Two transcripts of the ZM401 gene were detected by Northern
blotting analysis (1.2 kb, 2.0 kb). Southern hybridisation showed that the ZM401 in corn was present in one
or a very few copes in the maize genome.
Key words: Zea mays ; pollen; non-coding RNA; 5 RACE; 3 RACE; overlapping PCR; developmental
expression pattern
There were several reports of transcripts without a long
open reading frame (ORF) in various eucaryotes (Brannan
et al., 1990; Brockdorff et al., 1992; Brown et al. 1992; Askew
et al., 1994; Crespi et al., 1994; Velleca et al., 1994; Watanabe
and Yamamoto, 1994; Yoshida et al., 1994), and it has been
suggested that they function without being translated into
proteins. Some genes encode RNAs, rather than proteins,
as their final products. tRNA, rRNA, and the small nuclear
RNAs and nucleolar RNAs have been studied extensively,
and were relatively straightforward to identify by homol-
ogy searches or with specialized algorithms (Lowe and
Eddy, 1997; 1999). It has become apparent recently that in
addition to these structural RNAs, other mRNA-like non-
coding RNAs (ncRNAs) exist, which lack protein -coding
capacity and exert their action mainly or exclusively at the
RNA level (Eddy, 1999; Erdmann et a l., 1999; 2000; 2001;
Caprara and Nilsen, 2000; Storz, 2002). Analyses of the prop-
erties and functions of ncRNAs indicated that they could
act as gene regulators, as part of biotic and abiotic stress
signals, or as part of RNA-protein complexes with various
enzymat ic and structu ral activities. A number of ncRNAs
were processed in an mRNA-like manner. Consequently,
they undergo sp licing and have po ly(A) tails and ,
presumably, caps (Erdmann et a l., 2001). The presence of
ncRNAs has been described in several systems, for example,
in prokaryotic and eukaryotic sys tems (Willard and Salz,
1997; Panning and Janeisch, 1998; Akhtar et al., 2000).
Only a few ncRNAs from plants have been reported.
One of the first transcripts identified as an ncRNA in plants
was CR20 (Teramoto et al ., 1996), a gene iso lated from
cucumber (Cucumis satirus) that repressed by cytokinins
and by stress or developmental conditions (Teramoto et
al., 1995). This gene was part of a family o f ncRNA with
members in several plant species (Taylor and Green, 1995;
Teramoto et a l., 1996; van Hoof et a l., 1997). GUT15 was
ano ther characterized member o f this family (Taylor and
Green, 1995). The fact that transcripts o f this family were
hormonally regu lated and had unstable t ranscripts s ug-
gested that they might play a role in regulatory processes,
although their true functions were unknown. Another in-
teresting family of ncRNAs present in plants was typified
by Mt4 in Medicago truncatula (Burleigh and Harrison,
1998) and TPSI1 in tomato (Lycopersicon esculentum) (Liu
et al., 1997). As with the GUT15/CR20 family, these genes
were regulated by biotic (cytokinins) and abiotic (phosphate
starvation) signals. Several short non-conserved ORFs were
presen t in Mt4/TPS11 family, and all o f the transcrip ts
showed regions of absolute identity at the nucleotide level
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004498
(Mart in et al ., 2000). The high degree of nucleot ide se-
quence conservation and low level of ORF conservation
suggested that the final product of these genes was RNA
and not protein.
We previously isolated some cDNAs for pollen-s pe-
cific genes in Z. mays by differential screening (Li et a l.,
2001). One o f cDNA fragments, named ZM401, was s e-
lected to be analyzed. The length of ZM401 cDNA frag-
ment was 663 bp. Two trans crip ts (1.2 kb, 2.0 kb) of the
ZM401 were detected by Northern blot ting analysis. In
this study, we began our molecular analysis of ZM401 by
using 5 RACE and 3 RACE to clone a full-length cDNA for
the 1.2 kb RNA. We dis cuss the pos sibility that ZM401
was a non-coding RNA. Using RT-PCR and Northern blot-
ting analysis, we studied the developmental expres s pat-
tern of ZM401 in Z. mays pollen.
1 Materials and Methods
1.1 Plant material
Corn (Zea mays L.) po llen and anthers from various
stages of microspore development were collected from field
grown plants of the cultivar (Nongda 108). These were im-
mediately frozen in liquid N2 and stored at –70 ℃ until used
for RNA isolation.
1.2 3 and 5 RACE-based cloning of ZM401 cDNA
To isolate a full-length cDNA from total RNA extracted
from Z. mays pollen, we employed 3 RACE and 5 RACE.
ZM401-specific primers were designed based on the ZM401
cDNA fragments, to amplify the 3 end of ZM401 mRNA, an
anchored oligo(dT) primed single-strand cDNA was syn-
thesized from 2 mg of total RNA using AMV reverse tran-
scriptase followed by PCR with an ZM401-specific primer
(NGSP2) 5-GGTGAGGCGTCAATTTATAGGG-3 and an
anchor primer according to the manufacturer’s protocol (3
RACE System For Rapid Amplification of cDNA Ends, Ver-
sion 2.0, Gibco, BRL No. 18373-019). 5 RACE was performed
according to the manufacture’s protocol (5 RACE System
For Rapid Amplification of cDNA Ends, Version 2.0,Gibco,
BRL NO. 18374-058). In brief, single-strand cDNA was gen-
erated by revers e trans crip tion with a primer GSP1: 5 -
TAGCCATACTCCGGTGAC-3 . A homopolymeric dC tail
was added to the 5-end o f the cDNA by terminal
deoxynucleotidyl transferase followed by PCR with a nested
primer, GSP4: 5 -CTCACCGCCGTAAGCTCAATCTTGC-3,
and an abridged anchor primer was included in the kit. PCR
was performed under conditions of 30 cycles of 1 min at 94
℃, 1 min at 64 ℃ and 1 min at 72 ℃ followed by 10 min at
72 ℃ in a thermal cycle. Amplified PCR fragments were
cloned in to pMD18-T and s equenced . ZCF1 (5-
TTGCGAGCACATGGACGGAGGCGC-3 ) and ZCF2 (5 -
TTTTTTTTTTTTTTTGATCACGAACCTTATAATAAAGTTGAAGCTGG-
3 ) primers were designed for splicing 5 RACE and 3 RACE
amplified product.
1.3 Isolation of total RNA and reverse transcription-
polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from mature pollen and anthers
of various stages of microspore development，RNA was
iso lated us ing the ho t pheno l method，and subse-
quen tly treated with RNase-free DNaseⅠ(Promega Co.
Ltd.). Total RNAs (1 µg) were reverse-transcribed by AMV
reverse transcriptase (Promega Co. Ltd.) using oligo(dT)12-18
as a primer followed by PCR with ZM401-specific internal
primers (forward, P1 5 -ATCGGGAGCGAGTGGTGC-3 and
reverse P3 5 -CGTCACATGAAAACCGAAGAAC-3). PCR
react ion cond itions were as follows: 94 ℃ 1 min , 56 ℃ 1
min , 72 ℃ 1 min , 72 ℃ extension 10 min, 30 cycle. PCR
products were separated by electrophoresis in a 0.8% aga-
ros e gel fo llowed by b lo t ting to a ny lon membrane
(Amersham Pharmacia Biotech). The ZM401 products were
detected by hybridization with a 32P-labeled ZM401 cDNA.
In control experiments, RNA was treated as above but with-
out revers e transcriptase. In all cases, these con trol reac-
tions gave no signal after hybridization. All the RT-PCR
experiments presented in this study were repeated using
two or more independent RNA preparations.
1.4 Southern blot
Southern blotting analysis was performed as described
by Church method with slight modifications. Twenty mg
genomic DNA was digested with s ix restriction enzymes
(HindⅢ, EcoRⅠ, BamHⅠ, KpnⅠ, SacⅠ, EcoRⅤ), and
electrophoresis on an agarose gel, and transferred to a
Nylon membrane (Amersham Pharmacia Biotech) (Sambrook
et al., 1989). The membrane was prehybridized at 65 ℃ for
6 h in a prehybridization solution consisting of 1% BSA, 1
mmol/L EDTA, 0.5 mol/L Na2HPO4 (pH 7.2), 7% SDS. Probes
labelled with 32P were prepared from ZM401 cDNA using a
random-primed DNA labelling kit (Promega). Hybridization
wa s pe rfo r med with 32 p- lab ele d p r obe s in t he
prehybridization solution for 18 h at 65 ℃. The membrane
was washed at 65 ℃ in 2×SSC plus 0.5% SDS for 30 min,
1× SSC plus 0.5% SDS for 30 min , 0.5× SSC plus 0.1%
SDS for 30 min, and then, 0.1×SSC plus 0.1% SDS for 30
min.
1.5 Northern blot
Total RNAs (20 mg) from different stages of microspore
development and mature pollen g rains were isolated and
resolved by electrophoresis on a 1.2% agarose-formalde-
hyde gel. The RNAs were t ransformed to a ny lon




DAI Xiao-Yan et al.: Non-coding RNA for ZM401, a Pollen-specific Gene of Zea mays 503
1997). Although the transcripts of most of them, including
H19 , XIST, CR20 , wer e thought to funct ion as
untranslatable RNAs, the molecular mechanisms of their
funct ion still remain unclear. Watanabe and Yamamoto
(1994) demonstrated that mei RNA contained no long ORF
and formed a complex with Mei2 protein and performed an
essential role in the induction of meiosis in fission yeast.
The amino acid sequences deduced from all possible read-
ing frames of the ZM401 cDNA had many termination
codons throughout each reading frame, and no long ORF
was found, but it had poly(A) tail. The nucleotide sequence
showed no s ignifican t homology to non-coding mRNA
sequence isolated from plants. Therefore the ZM401 RNA
could be a member of a new category of non-coding RNA.
Northern blotting analysis revealed ZM401 was pollen-
specific gene (Li et al., 2001) and had two transcripts in Z.
mays mature pollen. RT-PCR and Northern blotting analy-
sis showed ZM401 began to express from tetrad stage of
microspore development to mature pollen. Northern blot-
ting analysis revealed the presence of two transcripts (2.0
kb and 1.2 kb) of ZM401 in Z. mays. There would be little
con tribution of degradat ion p roducts generated during
preparation of RNA to the observed two RNA bands, as
indicated by the fact that degradation of rRNAs was not
detected, the results could be due to (1) different members
of a gene family, (2) alternative splicing, (3) degradation of
the RNAs in the in tact plants, and (4) different transcrip-
tion start site. Genomic Southern blotting analysis showed
that ZM401 was encoded by a single or a very few copies
(Fig.9). Because there were at least two bands of ZM401
transcripts on Northern blots, and d ifferent patterns of
bands were obtained from the different stages of microspore
development, the hypothesis that a complex gene family
existed and/or degradation of the RNAs in the intact plants
occured failed adequately to explain the results. Therefore,
two transcripts o f ZM401 might derive from alternat ive
splicing of ZM401 gene or different transcription start site
if a single copy gene encodes ZM401 cDNA. If a few genes
encode ZM401, two trans cripts might derive from homol-
ogy gene of ZM401. Multiple transcripts, as in the case of
ZM401, were generated by alternative splicing for human
XIST (not in case of mouse) and cucumber CR20 (Brown et
al., 1992; Hao et al ., 1993), unstable mRNA generating
mult iple t ranscripts for GUT15 (MacIntos h et a l., 2001).
However, the significance of multiple transcripts for many
non-coding genes remains unknown.
Ecotop ic expression of ZM401 in tobacco or over-ex-
pression in maize all res ulted in pollen sterile (data not
shown), suggesting that ZM401 functions as a non-coding
mRNA and plays an important role in pollen development
of Z. mays.
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