全 文 ：高表达拟南芥 miR396 提高烟草抗旱性?
杨凤玺1 ,2 , 余迪求1??
(1 中国科学院西双版纳热带植物园 , 云南 昆明 650223 ; 2 中国科学院研究生院 , 北京 100049)
摘要 : MiR396 是一个由 21 个核苷酸组成的单链非编码 RNA 小分子。烟草内的 miR396 受干旱诱导说明其
可能参与烟草的干旱应答。在 35S强启动子作用下我们将 miR396 转入到烟草体内获得高表达转基因植株 ,
生理学测试表明高表达 miR396 的转基因烟草耐旱性增强 , 同时叶片表现出比野生型较低的失水率和较高
的相对含水量 , 进一步分析表明转基因植株除了叶片变得更为窄小外 , 其气孔密度和气孔系数都比野生型
降低 , 这些都表明 miR396 作为一个正调节因子参与烟草的干旱胁迫应答。
关键词 : miR396; 烟草 ; 干旱 ; 气孔
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 05 - 421 - 06
Overexpression of Arabidopsis MiR396 Enhances Drought
Tolerance in Transgenic Tobacco Plants
YANG Feng-Xi
1 , 2
, YU Di-Qiu
1 * *
( 1 Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223 , China;
2 GraduateUniversity of Chinese Academy of Sciences, Beijing 100049 , China)
Abstract: MiR396 was asingle-strandednoncoding small RNA with21 nucleotides, and theexpression of MiR369 in leav-
eswas strongly induced in water deficit condition in tobacco, which suggested a possible role of miR396 in drought stress
response . Under thecontrol of 35S promoter, MIR396 was introduced into tobaccomediatedby Agrobacteriumtumefaciens .
Physiological tests indicated that the elevated levels of miR396 increased drought tolerance in tobacco accompanying with
lower water loss rateand higher relativewater content . Further moremiR396-overexpressing plants exhibited visible reduc-
tions both in stomatal density and stomatal index aswell as a narrowand small leaf phenotype in comparison with wild-type
plants . The present study indicated that miR396 was a positive regulator in response to drought stress in tobacco .
Key words: miR396; Tobacco; Drought; Stomata
Water deficit is oneof themost universal environ-
mental stresses that affect almost all plant functions
(Zhu, 2001 ) . When water supply is limited, in order
to survive under such unfavorable growth condition,
plants have evolved diversemechanisms for adapting to
drought challenge ( Flexas and Medrano, 2002 ) . In-
creasing evidence has indicated that the molecular trait
of genes has the potential toovercome lotsof limitations
in improving drought-tolerance in tobacco, such as
mannitol-1-phosphate dehydrogenase ( mtlD) ( Abebe
et al. , 2003; Karakas et al. , 1997) , beanie aldehyde
dehydrogenase ( BADH ) ( Holmstrom et al. , 1996 ) ,
pryroline-5-carboxylase ( P5C ) ( Kishor and Miao,
1995) and lateembryogenesis abundant protin ( LEA) )
云 南 植 物 研 究 2009 , 31 (5) : 421～426
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.09044
?
?? ?Author for correspondence; E-mail : ydq@ xtbg. ac. cn; Tel : 86 - 871 - 5178133; Fax: 86 - 871 - 5160916
Received date: 2009 - 03 - 09 , Accepted date: 2009 - 06 - 01
作者简介 : 杨凤玺 (1985 - ) 女 , 在读硕士研究生 , 主要从事植物基因功能分析。 ?
Foun ?dation items: National High Technology Research andDevelopment Programof China ( 863 Program) ( 2006AA02Z129) , National Natural Sci-
ence Foundation of China (90408022) , Science Foundation of Yunnan Province (2004C0051M) , and“Hundred talents”Program of the Chi-
nese Academy of Sciences
(Zhang et al. , 2000) . Although the physiological and
molecular basis of bio-water saving or water uptaking
were complicated and diverse ( Chaves and Oliveira,
2004) , the control of water loss by stomata has, in
particular, received attention since plants loss more
than 90% of the water through the stomatal pore, and
stomata characteristics arehighly linked to drought tol-
erance (Flexas and Medrano, 2002 ) .
Recently, strong evidence suggested that miR-
NAs, which was a class of about 22nt, single-stranded
RNAs, regulatinggenes expression by targetingmRNAs
for cleavage or transcriptional repression ( Juarez et
al. , 2004; Chen, 2004; Griffiths-Jones et al. ,
2006) , are hypersensitive to abiotic or biotic stress as
well as to diverse physiological process ( Sunkar and
Zhu 2004; Lu et al. , 2005; Jones-Rhoades et al. ,
2006; Navaro et al. , 2006 ) . In Arabidopsis, miR398
was found to target CSD1 and CSD2 to improve toler-
ance of plant under oxidative stress conditions (Sunkar
et al. , 2006 ) . MiR395 and miR399 were identified to
be involved in sulfate and inorganic phosphate starva-
tion responses, respectively ( Jone-Rhoades and Bartel
2004; Fujii et al. , 2005; Kawashima et al. , 2009 ) .
Additionally, miR169 was investigated to function in
drought resistance as a negative regulator by targeting
NFYA5 in Arabidopsis (Li et al. , 2008) .
In this study, wefound that miR396 was marked-
ly induced by drought stress in tobacco . To further our
understanding of the roleof miR396 as a conserved se-
quence, we analyzed two transgenic plant lines over-
expressing AtMIR396a in tobacco ( Nicotiana tabacum
L .) showing that the increased levels of miR396 en-
hanced drought tolerance in tobacco . Meanwhile nar-
rower leaf and lower stomatal density than thoseof wild-
type were observed in transgenic tobacco, it suggested
that the reduced leaf size and the decreased number of
stomatal pore might partly make a contribution to the
enhanced drought resistance in tobacco .
Materials and methods
Plant materials and treatments Tobacco seeds ( Nic-
otiana tabacumCV . Xanthi NC) were surface sterilized, seeded
in culture dish containing MS medium, solidified with 0 .65%
(w?v) agar . After two weeks, the seedlings then transferred into
the soil in agreenhouse at 24℃- 28℃ . For PEG treatments, a-
week-old tobacco seedlingswere transferred into half-strength MS
solution after germination; the nutrient solution in the plastic
growth containers was continuously aerated with pumps and re-
newed everyweek . For drought treatment, 8-week-old seedlings
grown in soil were held withoutwater for 3 weeks before the pla-
ntswere rewatered . For ABA , GA , and NAA treatment, samples
of 8-week-old seedlingswere exposed to100μmol L - 1 ABA, 100
μmol L - 1 NAA, and1μmol L - 1 GA for 4 hours, respectively .
Plasmid construction and tobacco transformation A
fragment of 544 bp ( containing miR396a precursor of 151 bp)
was amplified from Arabidopsis genomic DNA by PCR using spe-
cific primers ( miR396a: 5′-TGC TGT AAA AGA ATG ACC
CTT-3′and 5′-AAA CTC ATA GAC AGA AGT TAG GGT T-
3′) , and then was inserted into a plant transformation vector
(pOCA30 ) , downstream of the constitutive 35S promoter . The
construct were introduced into tobacco by the leaf-disk method as
Curtis described ( Curtis et al. , 1995) mediated by Agrobacteri-
umtumefaciens strain GV3101 . Transgenic plants were screened
on MS mediumcontaining300μmL - 1 kanamycin . Two indepen-
dent lines of T2 plantswere used for detailed analysis .
RNA gel Blotting Total RNA was extracted from sam-
ples usingTrizol reagent ( Invitrogen) Samples of 10μgtotal RNA
were resolvedona15 % denaturing polyacrylamidee?1x TBE?7 M
ureagel and subjected to blot-hybridization analysis using [32 P]
ATP-labeled single-stranded anti-miR396a DNA probe ( CAGT-
TCAAGAAAGCTGTGGAA) , performed as described by Akber-
genov (Akbergenov et al. , 2006) .
Confocal Microscopy and Statistics To visualize sto-
mata outlines, the leaveswere immersed in0 .2 mgmL - 1 propid-
iumiodide for 30 min, with the blade adaxial side facing up . A
laser scanning confocal microscopeLSCM was used to take imag-
es . Statistics analysis followed the method described by Thomas
(Thomas et al. , 2003) .
Results
MiR396 Expression Induced by Water Deficit
MiR396 accumulatedmainly in leaves and flowers
and to a lower extent in roots and stems in tobacco
( unpublished data) . To study the expression patternof
miR396 in stress conditions, we detected the response
of miR396 expression to drought, ABA , GA and NAA
treatment by northern blot analysis . An obvious in-
crease in miR396 levels was observed when the seed-
lings were exposed to water deficit condition for 4 days
and the accumulation reached to thepeak at the day 7 ,
and then at the first day after re-watered miR396 re-
224 云 南 植 物 研 究 31 卷
turned to anormal level ( Fig. 1B) , whilenovisible in-
crease of miR396 was observed in wide-type plants
treatedwith ABA , GA or NAA ( Fig. 1C) . These re-
sults indicated that miR396 were up-regulated by
drought stress suggesting a potential role of miR396 in
the drought resistance in tobacco .
Fig . 1 Expression of miR396 in tobacco with drought, ABA , GA and
NAA treatment ( A ) Mature miR396 sequence . (B) Expression of miR396
in 8-week-old tobacco withheld fromwater for a week . ( C) Expression of
miR396 in leaves of 8-week-old seedlings exposed to 100μmol L - 1 ABA , 1
μmol L - 1 GA and 100μmol L - 1 NAA for 4 hours, respectively
MiR396 Overexpression Improved Drought Toler-
ance in Tobacco
The expression of miR396 induced by drought stress
in tobacco prompted us to determine whether miR396 was
involved in plant drought resistance . Given the miR396
was conserved in distantly related plant species, both in
terms of primary and mature miRNAs ( Zhang et al. ,
2006) , and miR396a differs from miR396b just by one
nucleotide ( Fig.1A ) , we generated transgenic tobacco
overexpressing AtMIR396 under the cauliflower mosaic
virus 35S promoter ( 35S: : AtMIR396a) . More than 10
transgenic lines were obtained and confirmed by PCR
analysis andnorthernblot annalysis, and thelines2 and 5
were selected for further studies (Fig.2) .
Fig . 2 Identification of 35S: : AtMIR396a plants by PCR ( A ) and
northern blot analysis using miR396a antisense probe ( B)
We utilized twotypes of water deficit conditions to
investigate the role of miR396 in drought stress re-
sponse . 8-week-old seedlings of transgenic and wild-
type plants were withheld fromwater for 3 weeks . The
wild-type plants leaves were clearly wilted compared to
those of transgenic plants ( Fig. 3A ) . In the parallel
experiments, PEG6000 (20% ) was used to produce a
more stringent water stress condition that did not per-
meate the roots but only imitated soil dehydration . The
leaves of wild-type plants wilted quickly upon 3 h
PEG6000 treatment, while those of the transgenic pla-
ntswere normal ( Fig. 3B ) . To determine whether the
response to osmoticumstress in seedling root was influ-
enced by miR396 , we analyzed root development and
germination rate under normal and drought conditions
(MS medium with 200 mmol L - 1 mannitol ) . Both of
themwere not significantly different between miR396-
overexpressing and wild-type seedlings ( data not
shown) , indicating that the modulation of miR396 in
tobacco drought stress tolerance was not associatedwith
the early stageof seedling development directly .
MiR396 Modulates the Drought Stress Response in
Tobacco Through Regulating Leaf and Stomata De-
velopment
To further elucidate the water stress resistance of
transgenic plants, we then detected daily water loss in
8-week-old transgenic and wild-type plants . As shown
in Figure 4 , water loss rates of leaves detached from
both two lines of transgenic plants were significantly
lower than that of wild-type plant ( Fig. 4A) . In addit-
ion, under both the well-watered condition and drought
stress condition, relative water contents ( RWC) of the
transgenic lines were higher than those of wild-type
samples and the distinction was more remarkableunder
3245 期 YANG and YU et al. : Overexpression of ArabidopsisMiR396 Enhances Drought Tolerance in . . .
Fig . 3 A and B Growth statuses of different transgenic lines under stress conditions . ( A ) 8-week-old seedlings wild-type (WT) and
two T2 transgenic plants ( line 2 and line 5) in greenhousewerewithheld fromwater for 3 weeks; ( B) 3-week-old seedlings were treat-
ed with 20 % PEG6000 for 3 hours; C-G . Measurement of stomatal number in wild-type and transgenic plants . C . gital images of im-
pressions from adaxial leaf surfaceof wide type; D and E . transgenic lines; analysisof stomatal density ( F) and index ( G) of abaxial
surfaceof mature leaves fromwild-type and transgenic plants . 8-week-old plants were used . ( P < 0 .05 )
water deficit conditions ( Fig. 4B) . These results sug-
gested that transgenic plants enhanced drought toler-
ance, at least partly, dueto an enhanced ability of bio-
water-savingwhichwas reported to bemainly controlled
by leaf area and stomata activity (Hill , 1994) .
Our previous study showed that miR396 accumu-
lation in tobacco led to a reduction in leaf size by tar-
geting three GRF-like genes ( unpublished data) ,
which might contribute to plants water-saving by de-
creasing transpiration ( Hill , 1994; Schoch et al. ,
1984) . In consistent with the small and narrow leaf ,
lower water-loss rate and higher relativewater content, a
lower stomatal densityand stomatal indexwereobserved
in transgenic plant . As shown in Figure 3 ( C-G) , the
stomatal density on the abaxial surfaceof the fifth leav-
es of 35S: : MIR396a transgenic line 2 and line 5 were
424 云 南 植 物 研 究 31 卷
Fig . 4 Effect of miR396 over expression on water loss and relative water content in wild-type and transgenic plants .
(A ) Water loss of the detached leaves from 8-week-old plants . (B) Measurement of leaf relative water content of 8-week-old
wild-type and transgenic plants at normal condition and water deficit condition ( a-week water withdrawal)
clearly decreased by 30% and 40% , respectively,
when compared with that of control plants grown under
the same condition ( Fig. 3E ) . Meanwhile an obvious
reduction in stomatal index: number of stornata related
to total epidermal cell number (Ticha, 1982) , in trans-
genic plantswasobserved (Fig. 3G) . All of theseresults
suggest that miR396 function as a positive regulator in
tobacco response to drought resistance .
Discussion
Drought is the primary limitation to plant produc-
tion worldwide, to cope with such unfavorable situ-
ation, most plants conserve water by reducing leaf size
and?or decreasing stomatal index ( Hill , 1994; Schoch
et al. , 1984; 1980) , since water conservation in pla-
nts are regulated to a large extent via the opening and
closureof stomata (Schurr et al. , 1992) . Stomata clo-
sure is the primary cause of the reduction in photosyn-
thetic rateundermild drought (Cornic, 2000; Medrano
et al. , 2002; Chen et al. , 1997) . Recent advances in
stomata development and the identificationof anumber
of genes regulating stomatal density make it possible to
generate transgenic plants with different stomatal densi-
ties (Xu and Zhou, 2008) .
In this work, drought-inducedmiR396 accumula-
tion has been detected, and the wild-type plants
showed sensitive physiological responses ( e.g ., leaf
rolling) to the sudden or continuous drought stress
(10% PEG treatment for 4 h or water-withheld for 3
weeks) , while no apparent external change was ob-
served in the transgenic plants ( Fig. 3 ) . Meanwhile,
we observed lower water loss rate and higher relative
water content in consistent with a lower stomatal density
and stomatal index compared to thewild-type plants as
well as small andnarrowleaves, while no distinction in
root development or germination rate in transgenic to-
bacco compared to that in the wild-type under normal
or drought stress . These data indicated that miR396
could improve drought tolerance in tobacco with en-
hanced ability of water conservation through reducing
leaf area and stomatal index but had no markedly influ-
ence in early stageof seedling growth . It also offered a
potential strategy to create drought-tolerant plants, for
miR396 was evolutionarily conserved both in terms of
primary andmaturemiRNAs and it would befeasibleto
obtain drought-tolerance transgenic plants by introduc-
ing 35S: miR396 into tobacco .
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