全 文 ：第 25卷　第 4期　　　　　　　　　　　　　植　　　物　　　研　　　究 2005年　10月
Vo .l 25　No. 4　　　　　　　　　　　　BULLETIN OF BOTAN ICAL RESEARCH Oc.t , 　2005
Foundat ion item:Supported by th e S tateKey Basic ResearchP roject(G19990160) and theKey Project of Ch ineseM inistry ofEducat ion (104191)
Au thor in troduction:Tang Zhonghua (1977— ), M ale, Assistan t, M ajor in p lan t secondary m etabolism andm olecu lar b iology.
*　Au thor for correspondence　E-m ai l:zygorl@pub lic. h r. h .l cn
Received date:2005 - 07 - 28
拟南芥MT-II过量表达提高抗旱性
唐中华　郭晓瑞　张洋洋　安志刚　于景华　祖元刚*
(东北林业大学森林植物生态学教育部重点实验室 , 哈尔滨　150040)
摘　要　富含巯基的植物 II型金属硫蛋白(MT)对植物抵抗重金属胁迫具有重要作用 ,其中一个
可能机制是金属硫蛋白可能猝灭重金属引起的氧化胁迫 。利用转MT-II基因和野生型拟南芥
(Arabidopsis tha liana)植株来对比研究 MT在胁迫过程中通过清除氧自由基 ,特别是 H2O2而对植
物抗旱性的影响 。研究表明 ,转基因型拟南芥能有效维持体内氧化 —还原势 ,减少 MDA的产生 ,
从而缓解干旱胁迫引起的伤害 ,提高抗旱性。
关键词　植物 II型金属硫蛋白;干旱抗性;氧化胁迫
Expression ofM eta llothionein-II inArabidopsis tha liana mi prove
desiccation tolerance
TANG Zhong-Hua　GUO X iao-Rui　ZHANG Yang-Yang　AN Zhi-G ang　YU Jing-Hua　ZU Yuan-Gang*
(Key Labo ra to ry of Fo rest E co logy, M inistry of Educa tion, Northea st Forestry University, H arb in　150040)
Abstract　M eta llo thione in-II(MT-II) has been identified to play an important role in the exposure of
plan t to heavy meta l like C d and Cu. O ne of themechanism s ofM T’ s proposed to p ro tec t plan ts during
th is process is tha t it can quench the reactive oxygen spec ies(ROS) activa ted by heavy-me tal exposu re.
He re w e demonstra ted the effect of expression ofMT-II in Arabidopsis thaliana on ox idative stress and
damage ex tent during drought stress. The RT-PCR resu lts suggested the imp lication o fMT-II against
drough t stress in the transgenic A rabidopsis. M easurements o fH2O 2 andma londia ldehyde (MDA) indi-
cated tha t ove r-expression o fMT-II in A. thaliana improved desiccation to lerance.
Key words　MT-II;desiccation to le rance;scaveng ing ROS
PlantMT-II like pro te in is a cyste ine rich po ly-
pep tide wh ich has incessantly been found in a range
of high plan ts. The func tions of p lantMT-II having
been understood so far are heavyme tals che lating and
detoxification
[ 1]
. Due to the mo re cyste ine-rich do-
ma in, Type 2 MT was more often used to be trans-
fo rmed into p lants to demonstrate the func tions in all
kinds of stress conditions. Recently, itw as suggested
thatMT-II may play a direct ro le ce llular defense a-
ga inst oxidative stress by functioning as antiox ida-
nts
[ 2, 3]
.
The produc tion of reactive oxygen species
(ROS) such as hydrogen pe roxide (H 2O 2) and su-
peroxide occurs at all times du ring p lant grow th and
deve lopment, bu t is increased when plan ts are ex-
posed to various bio tic and ab iotic stresses
[ 4 ～ 6]
.
These toxic ROS oxidize pro teins, fatty acids and
DNA , resu lting in cellular damage and ce ll dea th[ 7] .
P lants have produced a line o f mechanism s to re-
sponse to oxidative stress. In this paper, we used
transgenic A. tha liana seedlings to demonstrate the
ro le ofMT-II played in response to ox idative stress
caused by drough t condition.
1　Materials and methods
1. 1　DNA constructs and plan t transform ation
The plantMT-II like cDNA clone gene (see be-
low the sequence) iso la ted from B rassica Juncea was
constructed into the pETM 20 , an IPTG inducib le vec-
to r, and expressed as a fusion to TrxA (Thioredox in
A) prote in. Full-leng th B rassica Juncea MT-II cD-
NA sw e re iso lated as described p reviously. Transgen-
ic A. tha liana expressing the H is-taggedMT-II from
the CaMV 35S promoter ( in the b inary vector
pB I101) was generated using Agrobacterium tumefa-
ciens as described. T ransgenic A. tha liana plants o-
ve r-expression fo rMT-II were iden tified a fte r the in-
troduction o f a MT-II construct in pBI101. P lants
w ere grown in a greenhouse supplied w ith charcoal-fil-
tered air and we re used when 13 to 15 w eeks o ld.
Seeds of A. tha liana are w ild-type (eco type
“Columb ia”) and transgenic w ith MT. Bo th we re
sown in trays of pe rlite in tw o chambers respectively,
w ith photosynthe tic active radiation (PAR) o f 400
μmo l m - 2 s-1 , tempera ture o f 18℃ /25℃ in co r-
respondence to the pho toperiod of 10 h /14 h(nigh t /
day), 70% RH , and w atered w ith theHog land solu-
tion every o ther day.
Bo th kinds o f plan tsw ere treated w ith 30% po ly-
ethy lene g lyco l6000 (PEG ‘6000’ )when they f low-
ered, fo r during this period, p lant has the potential
capacity to w ithstand the ex ternal stress. A fte r being
incubated in so lution o f o smo ticum 2, 3, 4, 6, 8,
10 h , the plan tw as taken from the tray random ly.
1. 2　RT-PCR analysis
Tota l nucleic acid w as isola ted from whole leaves
and guard ce llp ro toplasts, and the DNA was removed
using RQ1 RN ase free DNase I (Promega, M adison,
W I). The absence of DNA in the samples w as con-
firmed after sa tu ra ting PCR (38 cyc les) w ith actin-
specific primers. One m icrog ram of tota l RNA was
used for cDNA synthesis using Omniscrip t RT (Q ia-
gen, Va lencia, CA)w ith o ligo (dT) 20 as the prim-
er.
1. 3　MDA con tent measurem ent using the w ay of
TBARS assays
M a londia ldehyde (MDA ) was ex tracted in
T ris-HC l buffer (20 mmo l L -1 , pH 7. 4). The ho-
mogena tew as cen trifuged at 2 500 g fo r 10 m in. The
MDA content w as measured in the supernatant w ith
the B ioxy tech K it LPO - 586 (Boeringer).
1. 4　H 2O 2 con tentmeasurement
TheH 2O 2 leve lw as colorime trically measured as
described by Okuda et a l
[ 8]
. H2O2 was ex tracted by
homogenizing 0. 5 g lea f tissue w ith 4mL of pe rchloric
acid (200mmol L - 1). The homogena tew as centri-
fuged a t 12 000 g for 10 m in and the supe rnatan t pu-
rified on Dow ex AG1-X2 before H 2O 2 measuremen.t
TheH 2O 2 leve lw as extrac ted and determ ined acco rd-
ing to Chaitanya and Na ithani
[ 9]
.
2　Results
2. 1　 Expression o fMT-II mRNA during drought
stress
RT-PCR determ ina tion of w ild type (WT) and
MT-II transgenic type (MT ) A. tha liana during
drought course w as used to demonstra te the MT-II
mRNA expression situation. The resu lts show ed tha t
F ig. 1　Expression ofMT-II mRNA inMT-II-transgenic-type and w ild-type A. tha liana seedling s
416 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
WT seedlings after de siccation d id not appear the ex-
pression o fMT-II transcripts, whe reasMT-II expres-
sion varia tion w as observed in MT seedlings after in-
cubation to drought condition and the peak expression
appea red at 1 h after incuba tion (Fig. 1). In contrast
to untrea ted contro l, MT-II transcrip t increase 8-fo ld
at the first stage o f desicca tion and then the decrease
of expression w as obse rved.
2. 2　MDA contents during drought stress
TheMDA content of transgenic controlw asmuch
highe r than that of the w ild-type contro.l On the con-
trary, a t the end o f the stress treatment, the MDA
conten t o f w ild-type w as much highe r than tha t o f
transgenic. For the transgenic, during the who le
course o f the stress trea tment, the MDA content
show ed unregu lar changes, and the changes we re
m ildly. A t the end of the stress treatment, the MDA
conten tw as much low er than that o f its con tro.l For
the w ild-type, a lso during the who le course of the
stress treatment, theMDA con tent show ed cu rv ilinear
changes, and this kind o f change w as much greater
than tha t of the transgenic. A t the end of the stress
treatment, the MDA conten t w as much higher than
that of its contro l, and the valuew as a lso a little h igh-
er than that of the transgenic contro l(Fig. 2).
F ig. 2　MDA changes inMT-II-transgenic-type and
w ild-type A. thaliana seedlings
2. 3　Changes o f endogenous H2O 2 during drought
stress
Endogenous H2O2 leve l is a significant indicator
of ox idative-reduced sta tus of p lants in a llk inds of en-
vironmen ts. H2O2 contents in M T and WT seedling s
we re measured in the present wo rk. The results sug-
gested tha t drought stress led to an obv ious decrease
be fore 1 h trea tmen t, subsequently 2-fo ld elevation of
H 2O 2 concen tration in WT seedling leaves. The in-
duced variation of endogenousH2O2 leve l observed by
drought stressw as no t strong like that ofWT seed ling
and the re w as just a slight reduction discovered (Fig.
3).
F ig. 3　H2O2 con tent change s inMT-II-transgen ic-type and
w ild-type A. thaliana seed lings
3　Discussion
P lantMT-II p ro tein has been shown to be imp li-
cated w ith heavy meta l tolerance, bu t themechanism s
rem ain to be discovered. Recent studies suggest that
itmay take part in the ce llu la r ROS scaveng ing and
a llevia te the damage due to exposure to heavy me tal
like copper
[ 3, 6]
. RT-PCR resu lts demonstrated in
presen t pape r showed that the re w ere noMT-II tran-
scrip ts in contro l and desiccatedWT seed ling leaves.
Th is w as supported by previousw ork tha t there no ex-
pression o fMT-II in A. thaliana seed ling s during de-
velopmen t and stress cond ition
[ 2, 6]
. A t the same
time, MT-II transcript variationsw ere observed inM T
seedling leaves during drought stress, indicating that
this pro te in w as involved w ith desiccation to le rance of
MT seed lings.
It has been suggested thatMDA leve l reflects im-
perfectly the loss ofmemb rane in tegrity
[ 10]
. M DA lev-
e ls inWT and MT A. thaliana seed ling leaves w ere
investiga ted to highlight the different to lerance of
4174期 唐中华等:拟南芥 MT-II过量表达提高抗旱性
them. The resu lts revealed thatMDA con tents inMT
seedling leaves remained homeostasis and experienced
a g radua l promotion under the same condition of des-
iccation. It w as the strong ev idence to exp lain that
su ffe red damage ex tent caused inMT by drought w as
slighter than tha t inWT.
RapidH 2O 2 accumu lation as w e ll as o ther ROS
in p lants is often induced by drough t stress. M o re oxi-
dative status w ill lead to serial damage to p lant
ce ll
[ 1, 7]
. It is surp rising to find tha tH2O2 accumu la-
ted 2-fo ld inWT leaves during exposure tow a te r defi-
cit, while inM T leave s H2O2 kep t conside rab ly sta-
b le. These resu lts implied that over-expressedMT-II
pro tein w as an ticipated to be imp lica ted to desiccation
pro tection ofMT seed lings and improved the desicca-
tion to lerance. The mechanism s involved w ith w ith-
standing drought stress remains to be uncove red.
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