全 文 ：第 40卷 第 2期
2016年 3月
南京林业大学学报(自然科学版)
Journal of Nanjing Forestry University (Natural Sciences Edition)
Vol．40，No．2
Mar．，2016
doi:10．3969 / j．issn．1000－2006．2016．02．011
收稿日期:2015－02－07 修回日期:2015－11－03
基金项目:“十二五”国家科技支撑计划(2012BAD23B05);中国博士后基金项目(2014M560427);国家自然科学基金项目
(31000294) ;江苏省博士后基金项目(1401042C);江苏高校优势学科建设工程资助项目(PAPD)
第一作者:林树燕(lrx@ njfu．com．cn)，副教授。* 通信作者:丁雨龙(yld@ vip．163．com)，教授。
引文格式:林树燕，郑笑，张莉，等． 鹅毛竹花药发育的超微结构观察［J］． 南京林业大学学报(自然科学版)，2016，40(2) :65－70．
鹅毛竹花药发育的超微结构观察
林树燕1，2，郑 笑1，张 莉1，姜明云1，丁雨龙1，2*
(1．南京林业大学南方现代林业协同创新中心，南京林业大学生物与环境学院，江苏 南京 210037;
2．南京林业大学竹类研究所，江苏 南京 210037)
摘要:以鹅毛竹为材料，采用透射电子显微镜技术对花药发育的超微结构进行了系统研究。结果表明:鹅毛竹花
药具 4个药室，花药壁由表皮、药室内壁、中层和绒毡层组成。花药壁发育为单子叶型。造孢时期的造孢细胞胞
质较浓厚，线粒体、高尔基体丰富，含有大量的小泡，有少量油滴;小孢子母细胞分裂期超微结构特征明显，分裂
前期小孢子母细胞内含大量小泡、环状片层、高尔基体及线粒体，分裂期的小孢子母细胞胞质中的小泡、线粒体
更加丰富，高电子致密颗粒的数量明显增加;二分体细胞内具有丰富的线粒体，个别细胞壁上有胞质通道，胼胝
质壁加厚;刚释放出的小孢子壁薄，线粒体、环状片层丰富;中央期小孢子形成期呈圆球形，细胞质稠密，具有丰
富的线粒体、内质网、高尔基体等细胞器。随着小孢子的发育，其外壁逐渐增厚，在质膜外侧形成了 3 条宽窄不
等的电子致密带，即外壁表层、基粒棒层和外壁内层，此时形成萌发孔;成熟花粉粒充满淀粉粒。在花药发育期
间出现雄性败育现象，主要表现为绒毡层细胞过早解体和小孢子母细胞及单核小孢子形态畸形，这些异常是雄
性败育的主要原因之一。
关键词:鹅毛竹;花药发育;超微结构;花粉;竹亚科
中图分类号:S718 文献标志码:A 文章编号:1000－2006(2016)02－0065－06
The ultrastructure of anther development in Shibataea chinensis Nakai (Bambusoideae)
LIN Shuyan1，2，ZHENG Xiao1，ZHANG Li1，JIANG Mingyun1，DING Yulong1，2*
(1． Co-Innovation Center for the Sustainable Forestry in Southern China，College of Biology and the
Environment，Nanjing Forestry University，Nanjing 210037，China;2． Bamboo Ｒesearch Institute，
Nanjing Forestry University，Nanjing 210037，China)
Abstract:Shibataea chinensis belongs to the Bambusoideae，a large plant family with more than 1 000 bamboo species．
Lacking of normal pollen development is one of the bottlenecks of bamboo molecular breeding． In order to provide addi-
tional data on this blossomed bamboo family，the ultrastructure of anther of S． chinensis was investigated with the methods
of TEM in this study． The results are as follows:The anther wall consists of four layers． They are epidermis，endotheci-
um，middle layer and tapetum． The development of anther wall belongs to the monocotyledonous type． The secondary
sporogenous cells have some vesicles，annulate lamella，Golgi bodies and mitochondria． There are a plenty of vesicles，
mitochondria and black high electron-dense cytoplasm in microspore mother cells． Dyad produces after the first meiosis，
there are cytoplasmic channels on the cell wall with the thick callus． Pollen mother cells undergo the second meiosis to
give rise to tetrad． Three electron-dense bands (the outer wall surface，the grana layer and the endexine)develops out-
side the pollen cell membrane during later central nucleus microspore，and the pollen germination aperture also develops
at this time． When the pollens mature，many white plasmids and starch grains are in them． Male sterility phenomenon oc-
curs during the process of anther development，such as the phenomena that the tapetum disintegrated earlier，the pollen
mother cells and microspores are abnormal． So the abnormality of pollen is associated with the abortion of microsporocyte
and microspore．
Keywords:Shibataea chinensis;anther development;ultrastructure;pollen;Bambusoideae
南 京 林 业 大 学 学 报 (自 然 科 学 版 ) 第 40卷
1 Introduction
The family Bambusoideae comprises more than
1 000 species，mainly distribute in subtropical and
tropical regions． Bamboo is one of the most important
forest resources in the world． It mainly distributes in
Asia，Africa and Latin America． People used bamboo
for various purposes such as food，building materials，
interior decoration materials， papermaking，
landscaping and so on． Our understanding of bamboo
has been considerably enhanced recently on the basis
of previous researches［1－5］．
Bamboo plants are generally characterized as
having a relatively long vegetative growth period，usu-
ally from three years to one hundred years，and once
it blossoms，either the whole population or the blosso-
ming individuals will die off． So it is necessary and
urgent to study the reproductive characteristics once
bamboo blossoms． Some bamboos will produce large
number of fertile caryopsis after blossoming， like
Phyllostachys edulis，Sasa sinica，Fargesia murielae，
Chusquea culeou． However，some species never bear
fruit， like Phyllosatchys bambusoides， Shibatea
chinensis，or only very little fruits were available． So it
is not easy having an opportunity to study the embry-
onic development of bamboo plants［6］． Compared with
its various application researches，there are only a few
reports about its fundamental biological research，es-
pecially on its reproductive biology． As so far works
on general embryological descriptions have focused on
a limited number of bamboo species，such as Phyl-
lostachys edulis［7］， Chimonobambusa marmorea［8］，
Drepanostachyum microphyllum［9］， Phyllostachys
praecox［10］，Dendrocalamus sinicus［11］，Menstruocala-
mus sichuanensis［12］． However there is no contribution
concerning the ultrastructure of microspores during
microsporogenesis in the family Bambusoideae．
Shibataea chinensis is an excellent ornamental
bamboo species，and it was widely cultivated in Jian-
gsu，Jiangxi，Fujian，Anhui Province of China． In
recent years，it has been blossoming in the Bamboo
Garden of Nanjing Forestry University，and no seed
were found，which provides a precious opportunity on
embryology research．
The ultrastructure of anther in S． chinensis has
not previously been investigated． In order to find
whether the sterility is caused by the abnormal pollen
development or not，this study offers a partial remedy
to this situation by providing an account of anther
wall， microspore and male gametogenesis
development in S． chinensis．
2 Materials and methods
The flower buds and blossoming flowers of S．
chinensis were collected from Octomber 2008 to April
2011． They were planted in bamboo garden of Nanjing
Forestry University，Jiangsu Province，China．
Anthers for transmission electron microscopy
(TEM)were collected from fresh flowers，which were
fixed in 4% paraformaldehyde and 2. 5% glutaralde-
hyde (0. 1 mol /L phosphate buffer，pH 7. 2)imme-
diately，and air was removed under vacuum，at 4 ℃
24－48 hours． Then the anthers were washed in 0. 1
mol /L phosphate buffer (pH 7. 2)with 1% osmium
tetroxide (0. 1 mol /L phosphate buffer，pH 7. 2)
overnight at 4 ℃，washed in phosphate buffer，and
then dehydrated in an ethanol series，acetone transi-
tion． Anthers were embedded in Epon 812 resin． Sec-
tions were cut with a LKB－V rotary ultramicrotome，
stained with uranyl acetate and lead citrate，and ob-
served with an H － 600 transmission electron micro-
scope．
3 Ｒesults
3．1 Formation of anther wall
The anther of S． chinensis is tetrasporangiate and
glandular tapetum． The anther wall consists of an epi-
dermis，an endothecium layer，a middle layer and a
tapetum． The development type of anther wall is mon-
ocotyledonous． When the pollen matures，the anther
wall is composed of the cutinization epidermis and a
highly fibrous，thickened endothecium．
The archesporial cell undergoes a periclinal divi-
sion，giving rise to parietal and primary sporogenous
tissues． The former divides periclinally and forms two
layers of cells． The outer of which produces an endo-
thecium． The inner layer divides again and gives rise
to a middle layer and a tapetal layer，which are the
characteristics of the“monocotyledonous type”．
At the stage of secondary sporogenous cells，ta-
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第 2期 林树燕，等:鹅毛竹花药发育的超微结构观察
petum cells are dense cytoplasm，and they are mostly
in meiosis (Fig．1a)． At the microsporocyte stage，the
pollen wall with four layers has developed (Fig．1b)．
There is no starch grains in the epidermis but a few
starch grains are in the endothecium (Fig．1c)． Tape-
tum cells enlarge greatly (Fig． 1d)and form a rich
endoplasmic reticulum slot (Fig． 1e)． During tetrad
formation，a lot of starch grains appear in epidermis
and endothecium (Fig．1f)．The middle layer becomes
flattened． Tapetal cells are elongated tangentially
(Fig． 1g)，usually having two or four nucleates，
which are rich in mitochondria， endoplasmic
reticulum and golgi (Fig．1h，1i)． At the stage of sys-
tolic microspore， some endoplasmic reticulum and
ubisch form in the tapetum cells (Fig． 1j)． At the
stage of central microspore，starch grains are in the
epidermis and endothecium (Fig． 1k) ，and the
middle layer and tapetal cells begin to degenerate，
where phagocytic vesicles come into being (Fig．1l)．
At the stage of late uninucleate microspore，the epi-
dermis cell is in cornification，the endothecium layer
are still rich in starch grains (Fig．1m) ，and the mid-
dle layer and tapetal cells degenerate gradually． At
the meiosis stage of mononuclear microspore， the
middle layer and tapetal cells further degrade，only
having a thin layer left． By the time the pollen grains
are mature，the cutinization is very obvious in the ep-
idermis cell (Fig．1n) ，the endothecium layer has de-
veloped highly fibrous thickening (Fig．1o) ，and the
tapetal cells and the middle layer has completely de-
generated．
a． at secondary sporogenous cell stage，showing the tapetum cell in the anther wall;b． at microspore mother cell stage，showing the
epidermis cell in the anther;c． showing the starch grains in the endothecium;d． showing the tapetum of anther;e． showing the endo-
plasmic reticulum slot in the tapetum cells;f． anther wall at dyad stage，showing starch grains in the epidermis，and endothecium;g．
showing radial lengthened tapetal cells;h． tapetal cell，showing abundant mitochondria，endoplasmic reticulum，Golgi body;i． abun-
dant endoplasmic reticulum in tapetal cells;j． showing ubisch in tapetum;k． anther wall，showing abundant starch grains in the en-
dothecium，the middle layer degraded，but the wall still existed，tapetal cells begin to degradation;l． chromatin contract in tapetal
cells，empty vesicle appeared;m． monokayotic microspore with the nucleus located aside，showing abundant starch grains in endothe-
cium and some surviving tapetal cells;n． mature anther wall，showing outer tangential wall of epidermis cornification;o． mature an-
ther wall，showing endothecium fiber thickening．
Fig．1 The ultrastructure of the anther wall development in Shibataea chinensis
图 1 鹅毛竹花药壁发育超微结构
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南 京 林 业 大 学 学 报 (自 然 科 学 版 ) 第 40卷
3．2 Microsporogenesis
The epidermis cells and meristematic cells
arrange from outside to inside of young stamen． These
cells are small with dense cytoplasm，small vacuoles
and thin cell walls． An archesporial cell is larger than
other cells，which is beneath each corner of epidermal
cells． The archesporium is divided into a parietal cell
and a primary sporogenous cell by the periclinal divi-
sion． Then the primary sporogenous cells produce sec-
ondary sporogenous cells． Sporogenous cells are large，
arrange tightly and take the shape of irregular poly-
hedron，which have cytoplasm，abundant mitochon-
dria，Golgi bodies，vesicles and a small amount of oil
droplets (Fig．2a，2b，2c)． When the anther is 3 mm
long，the secondary sporogenous cells give rise to the
larger microspore mother cells，which have large nu-
cleus，no obvious vacuole and are surrounded by cal-
lose． There are some vesicles， annulate lamella，
Golgi bodies and mitochondria (Fig． 2d)． When the
anther is 5－6 mm long，the microspore mother cells
are in the process of meiosis． There are a plenty of
vesicles，mitochondria (Fig． 2e)，and black high
electron-dense cytoplasm(Fig．2f) ;Through prophase
I，metaphase I，anaphase I and telophase I，the first
meiotic produces dyad(Fig．2g) ，which has plenty of
mitochondria． There are cytoplasmic channels on the
cell walls with the thick callus． Pollen mother cells
undergo the second meiosis to give rise to tetrad． The
cytokinensis meiosis was successive and produced bi-
laterally symmetrical tetrads．
a． secondary sporogenous cell，showing abundant mitochondria in the cytoplasm;b． secondary sporogenous cell，showing little lipid in
the cell;c． at secondary sporogenous cell stage，showing abundant vesicles inside;d． showing abundant Golgi body and mitochondria in
microspore mother cell;e． at microspore mother cell prophase，showing abundant vesicle and mitochondria;f． showing the abundant ves-
icle and osmiophilic globule;g． showing mitochondria and some other organelles in the dyad;h． the stage of contraction of microspore，
showing abundant mitochondria and the annulate lamella;i． at the middle microspore stage，showing mitochondria，endoplasmic reticu-
lum and microspore wall;j． middle microspore stage，showing the formation of germinal aperture;k． showing monokayotic microspore
with the nucleus located aside;l． microspore with the nucleus located aside，showing golgi body and mitochondrial;m． two-cell pollen，
showing generative nucleus section and vegetative nucleus section;n． showing mature pollen wall;o． showing the shape of starch grains
in mature pollen．
Fig．2 The ultrastructure of the pollen development in S．chinensis
图 2 鹅毛竹花粉发育的超微结构
3．3 Male gametophyte development
When the anther length is 6 － 7 mm long，free
microspores are released into the pollen sac with the
dissolution of the callose walls． These free microspores
86
第 2期 林树燕，等:鹅毛竹花药发育的超微结构观察
are called systolic microspore with thin cell wall，a lot
of mitochondria，annulate lamella(Fig． 2h)and the
nucleus in the central． Later central nucleus
microspore takes on a dense cytoplasm form． At this
stage，three electron-dense bands develops outside
the pollen cell membrane，the outer wall surface，the
grana layer and the endexine(Fig．2i)，and the pollen
germination also develops at this time(Fig．2j)． When
the anther length is 8－9 mm long，the central vacuole
develops，the nucleus takes a peripheral position and
there are a few mitochondria and Golgi bodies in the
late uninucleate stage(Fig．2k，2l)． At this stage，the
outer wall of microspore germination holes is thicker
than before． When the anther length is 10 － 11 mm
long，the first mitotic division of the microspore nu-
cleus results in the formation of two unequal cells，a
larger vegetative one and a smaller generative one
(Fig． 2m)． There are many white plasmids and starch
grains in the pollen grains． The aperture is very clear
at this stage． The extine layer of the pollen wall is
thicker than the intine one(Fig．2n，2o)．
3．4 Abnormal phenomena in anther
Pollen abortion can cause low seed set，and S．
chinensis has no seeds． Some abortion and
irregularities occur in microsporocyte，meiosis and
during the free microspore stage． In some microspo-
rangia，an early degradation of the tapetum caused
the microsporocytes to degenerate at the
microsporocyte and tetrad stage (Fig． 3a，3b，3c) ，
which can result in the formation of a large cavity．
Sometimes when tapetum cells encounter hypertrophy，
they almost fill the microsporocyte． In other anthers
many abnormally shaped microsporocytes and micro-
spores were found in the development of pollen (Fig．
3d，3e，3f)．
4 Discussion
Anther wall development of S． chinensis is de-
scribed in detail for the first time in this study． Ac-
cording to the characteristics observed in S． chinensis，
anther wall development is the monocotyledonous
type，the inner secondary parietal layer divides to
give rise to the middle layer and the tapetum，which
is in accordance with David［13］． The anther wall con-
sists of an epidermis，an endothecium layer，a middle
a，b，c showing degenerated tapetum cells and microspores;d，e，
f showing deformational microspore．
Fig．3 The abortion during the development of anther
图 3 鹅毛竹花药发育过程中的异常
layer and a glandular tapetum． It has successive cyto-
kinensis meiosis and produces bilaterally symmetrical
tetrads． Mature pollen grains are 2 － celled with one
germinal aperture． The pollen is nearly spherical．
According to the formation of the middle layer，
Davis classified the development of anther walls into
four types:basic，dicotyledonous，monocotyledonous
and reduced［13］． The monocotyledonous type includes
the majority of monocotyledonous families as well as
several dicotyledonous families． The anther wall of S．
chinensis consists of four layers (epidermis，endothe-
cium，middle layer and tapetum)，which belong to
the monocotyledonous type． Meanwhile，the tapetum
was the typical glandular type，and the cytokinesis of
microsporocyte in meiosis was the successive type．
This finding is in agreement with that of Wang et
al．［11］ and Lin et al．［14］． At the same time，Huang et
al．［10］ pointed out that there were two rows of middle
layer in the anthers of Ph． praecox and one of the
middle layers degenerated and disappeared in subse-
quent development［14－15］．
Embryological studies are often useful not only in
the events relevant to sexual reproduction but also in
solving taxonomic and phylogenetic problems at or
above genetic levels［16－20］． In angiosperms，the ar-
rangement of microspores in a tetrad is recognized:
tetrahedral，isobilateral，linear，T － shaped and de-
cussate［13］． The tetrad patterns were particularly asso-
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南 京 林 业 大 学 学 报 (自 然 科 学 版 ) 第 40卷
ciated with the type of microsporogenesis． S． chinensis
is the isobilateral tetrad type，which was different
from that of Wang et al．［11］ and Huang et al．［10］． They
reported respectively that the microspore tetrads were
tetrahedral type in Dendrocalumus sinicus and Ph．
praecox［14］．
S． chinensis，unlike its close relative，moso bam-
boo，is reported to produce no seeds． In the present
study，we found that many microspores develop ab-
normally at the microsporocyte and tetrad stage and at
the early free microspore stage． According to Johri
etal［19］ and Ettore et al［21］，during the pollen devel-
opment，tapetum layer serves mostly as a tissue for
meiocyte / spore nutrition． In addition to this main
function，the tapetum has other functions，namely the
production of the locular fluid，the production and re-
lease of callose，the conveying of P．A．S． positive ma-
terial towards the loculus，the formation of exine pre-
cursors，viscin threads and orbicules (= Ubisch
bodies)，and the production of sporophytic proteins
and enzymes，and of pollenkitt / tryphine［14］． The de-
generation of the tapetum is responsible for the
abortion of the pollen［22］． In S． chinensis，the tapetum
cells began to degenerate at the premeiotic stage and
were absorbed completely in the development of the
microspores． Similar trends were reported［10，14］．
参考文献(reference):
［1］Peng Z H，Lu Y，Li L B，et al． The draft genome of the fast－
growing non-timber forest species moso bamboo (Phyllostachys
heterocycla)［J］． Nat Genet，2013，45:456－461．Doi:10．1038 /
ng．2569．
［2］Zhang X M，Zhao L，Larson-Ｒabin Z，et al． De novo sequencing
and characterization of the floral transcriptome of Dendrocalamus
latiflorus (Poaceae:Bambusoideae) ［J］． PloS One，2012，7:
e42082．Doi:10．1371 / journal．pone．0042082．
［3］Wei Q，Cao H，Li Z，Kuai B，et al． Identification of an AtCＲN1
－like chloroplast protein BeCＲN1 and its distinctive role in chlo-
rophyll breakdown during leaf senescence in bamboo (Bambusa
emeiensis‘Viridiflavus’) ［J］． Plant Cell，Tissue and Organ cul-
ture，2013，114:1－10．Doi:10．1007 /s11240－013－0298－y．
［4］Peng Z，Zhang C，Zhang Y，et al． Transcriptome sequencing and
analysis of the fast growing shoots of moso bamboo (Phyllostachys
edulis) ［J］． PloS One，2013，8:e78944．Doi:10．1371 / journal．
pone．0078944．
［5］Mudoi K D，Saikia S P，Goswami A，et al． Micropropagation of
important bamboos:a review［J］． African Journal of Biotechnolo-
gy，2013，12:2770－2785．Doi:10．5897 /AJB12．2122．
［6］Zhou F C． Bamboo cultivation［M］． Beijing:China Forestry Pub-
lishing House，1998．
［7］Qiao S Y，Liao G L． Embryology observation of Phyllostachys pu-
bescens［J］． Bamboo Ｒesearch，1984，3(1) :15－23．
［8］Hu C H，Yu F G，Pang Y J． Observation and Study on Embryolo-
gy of Chimonobambusa marmoreal ［J］． J Bamboo Ｒesearch，
1994，13(4) :6－11．
［9］Pang Y J，Yu F G，Hu C H． Preliminary observation on abnormal
development of the staments of Drepanostachyum microphyllum
［J］． J Bamboo Ｒesearch，1994，10(4) :42－46．
［10］Huang J Q，Huang H H，He F J． The formation of microspore and
the development of male gametophyte of Phyllostachys praecox
［J］． Journal of Bamboo Ｒesearch，1999，18(3) :55－58．Doi:10．
1677 /EＲC－10－0014．
［11］Wang S G，Pu X L，Ding Y L． The structures of reproductive or-
gans and development of the female and male gametophyte of Den-
drocalamus sinicus［J］． Bulletin of Bot Ｒesearch，2006，26(3) :
270－274．Doi:10．3969 / j．issn．1673－5102．2006．03．006．
［12］Lin S Y，Hao J J，Xin H． The megasporogenesis，microsporogen-
esis and the development of their female and male gametophyte in
Menstruocalamus sichuanensis［J］． Journal of Nanjing Forestry U-
niversity(Natural Sciences Edition) ，2009，33(3) :9－12．Doi:
10．3969 / j．issn．1000－2006．2009．03．003．
［13］Davis G L． Systematic embryology of the angiosperms［M］． New
York:John Wiley and Sons Inc，1966:528．
［14］Lin S Y，Ding Y L． Development of the male and female gameto-
phytes of Arundinaria simonii f． heterophylla［J］． Acta Botanica
Boreali-Occidentalia Sinica，2012，32(5) :907 － 914． Doi:10．
3969 / j．issn．1000－4025．2012．05．010．
［15］Lin S Y，Ding Y L． Observations on megasporogenesis，micros-
porogenesis and development of the male and female gametophytes
of Arundinaria simonii f． Heterophylla［J］． Scientia Silvae Sinicae．
2013，49(8) :168－175．Doi:10．11707 / j．1001－7488．20130824．
［16］Arias T，Williams J H． Embryology of Manekia naranjoana (Pip-
eraceae)and the origin of tetrasporic，16－nucleate female game-
tophytes in Piperales［J］． American Journal of Botany，2008，
95:272－285．Doi:10．3732 /ajb．95．3．272．
［17］Tobe H． The embryology of angoisperms:its broad application to
the systematic and evolutionary study ［J］． Botanical Magazine
(Tokyo) ，1989，102:351－367． Doi:10．1007 /BF02488572．
［18］Fang C，Wang Y X，Wang S S，et al． Molecular control of mi-
crosporogenesis in Arabidopsis［J］． Current Opinion in Plant Bi-
ology，2011，14:66－73．Doi:10．1016 / j．pbi．2010．11．001．
［19］Johri B M，Ambegaokar K B，Srivastava，P S． Comparative em-
bryology of angiosperms［M］． Berlin:Springer－Verlag，1992．
［20］Madrid E N，Friedman W E． emale gametophyte and early seed
development in Peperomia (Piperaceae) ［J］． American Journal
of Botany，2010，97:1－14．Doi:10．3732 /ajb．0800423．
［21］Ettore P，Franchi，Michael H． The tapetum:its form，function，
and possible phylogeny in embryophyta ［J］． Plant Systematics
and Evolution， 1985， 149 (3 － 4) :155 － 185． Doi:10．
1007 /BF00983304．
［22］Liang C L，Liu M J，Zhao J． Ｒesearch progress on plant seeds a-
bortion［J］． Molecular Plant Breeding，2005，3(1) :117－ 122．
Doi:10．3969 / j．issn．1672－416X．2005．01．020．
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