全 文 ：武汉植物学研究 2007，25(3)：222—225
Journa／of Wuhan Botanical Research
DREB类转录因子介导的烟草抗非生物胁迫特性研究
刘卫群 ， ，石永春 ，胡亚杰2，刘巧真2
(1．河南农业大学生命科学学院，郑州 450002；2．国家烟草栽培生理生化重点实验室，郑州 450002)
摘 要：检测并分析了转BnDREB1．5基因烟草叶片的持水性能，上、下表皮气孔大小和密度及叶绿素含量，并在自
然失水 7 h后检测了细胞的离子泄漏状况。结果表明：转基因烟草叶片单位时问内每平方厘米的失水量是野生型
烟草叶片的62％；上表皮的气孔大于野生型，而气孔开度小于野生型；野生型烟草叶片上的气孔密度接近转基因烟
草的 1．5倍 ；野生型烟草叶片的叶绿素含I1~转基因烟草叶片高 29％；野生型烟草叶片的质膜相对透性是转基因
烟草叶片的 1．4倍。
关键词 ：烟草；DREB转录因子；抗非生物胁迫 ；生物学性状
中图分类号：Q78；$572 文献标识码：A 文章编号：1000-470x(2007)03-0222-04
The Tolerance to Abiotic Stresses M ediated by DREB-like
Transcription Factors in Nicotiana tabacum
LIU Wei—Qun ，SHI Yong．Chun ，HU Ya-Jie ，LIU Qiao—Zhen
(1．1ife Science Colege，Henan Agricultural University，Zhengzhou 450002，China；
2．National研 Lab ofTobacco Culture andPhysiology andChem／stry，Zhengzhou 450002，China)
Abstract：The biological properties on tobacco with 35S：BnDREB1-5 transformation were analyzed which
included the holding water capacity，the size an d density of upper and lower epiderm is stomata，the con—
tent of chlorophyl，an d plasma membran es perm eability after physical drought stressed 7 h．The results in—
dicated that water—holding capacity of 35S：BnDREB1-5 tran sgenic tobacco leaves was 62％ of that of
wild—types．Th e upper epiderm is stomata apparatus was bigger in tran sgenic tobacco than wild—type，but
the opening of stoma was smaler in upper epiderm is than wild—types．Th e stomata density was 1．5 times
in wild—type leaves as large as in tran sgenic leaves．Th e content of chlorophyl was 29％ higher in leaves
of wild—type than in transgenic leaves．an d the plasma memb ranes perm eability were 1．4 times as much as
in tran sgenic tobacco．
Key words：Tobacco(Nicotiana tabacum)；DREB transcription factor；Abiotic streses；Biological
properties
Drought，high—salt and low—temperature are stres—
ses that cause adverse efects on growth an d develop—
ment of tobacco resetling stage an d result in ab loom
stage ahead，which bring tobacco farmers about serious
loss of economy．To deal with this problem ，tobacco sci—
entists have taken man y measures in plan ting and ferti—
lization technique for a long time，but that is idea1．Th at
is because the characters of plan t stress—resistance are
far more complicated compared with the correlated
characters of plan t insect·-resistance and disease·-resis·-
tance．Th e capability of plant to toleran ce of drought，
high—salt and cold—stress is independent of a single
functional gene．Th e character of plant stress—resistance
is influenced by many functional genes ．Th us，it is
important to enhan ce the control capab ility of one key
regulatory factor to make the plan t achieve ideal ，multi—
pie and functional improvement for stress—resistance ．
DREB (dehydration responsive element·binding
protein)transcription factor plays an important role in
regulating gene expression in that response to drought，
high—salt and cold—stress[。·引
． It may be a more efective
strategy to improve or enhance the control capab ility of
the key tran scription factor for the ideal an d multiple
efects compared with the conventional methods which
收稿日期：2006．11．o3，修回日期：2006·12-26。
基金项 目：河南省科技攻关项目(062405O012)资助。
作者简介：刘卫群(1958一)，博士，教授，博士生导师，从事烟草生化与分子生物学研究(E·mail：liuweiqun@eyou．corn)。
+ 通讯作者。
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第 3期 刘卫群等：DREB类转录因子介导的烟草抗非生物胁迫特性研究(英)
a single functional gene is trans~ ed for improving a
single trait．We transfe~ed the BnDREB1-5 from Bras—
sica napus to tobacco plant and investigated the abiotic
stress tolerance and some biological properties of trans—
genie tobacco leaves．
1 M atedals and methods
1．1 Materials
Transgenic tobacco with 35S：BnDREB1-5 was
used in this experiment．
1．2 M ethods
1．2．1 Leafwater loss(LWL)
The areas of leaves of transgenic and wild type to—
bacco(WT，hereinafter the same)were measured by
PLANT LEAF C1-202(made in U．S．A．)．Then leaf
water loss of in vitro transgenic an d wild type tobacco
was determined at room temperature an d weighed every
30 min for 7 h．Leaf water loss rate was calculated
．
1．2．2 The upper and lower epidermis stomata of
transgenic tobacco leaves
After being incubated on humid filter paper for
one hour，the upper and lower epiderm is were isolated
from the leaves an d then the stomata parameters f in—
eluding densi~， length ，width，etc．)in thatwere exam—
ined with a Olympus microscope．
1·2．3 The contents of chlorophyll
The method of it accord to reference[5]．
1·2．4 Th e plasma membrane pe rmeability
The method of it accord to reference[5]．
2 Results
2．1 Th e analysis ofleafwater loss
Th e rate of LWL was obviously slower in tran sge—
nic tobacco than wild type for 7 h(Fig．1)．In the first
hour，the rate of wild type is 1．62 times as much as it
4
0 3 4 5 6 7
1 e(h)
Fig·1 Analysis of LW L of transgenie tobacco
and wild type
of tran sgenic tobacco．And then the rate of tran sgenic
tobacco was lower than wild—types during all the treated
time．7 hours later，the rate of tran sgenic tobacco was
0．33 mg H2O／cm per hour，an d the rate of wild—types
was 0．54 mg H2 O／cm per hour．The amount of lost
water from transgenic tobacco leaves was 62％ as much
as it of wild—types．
2．2 The analysis of stomata parameters
Compared with wild types，the stomatal apparatus
of upper epiderm is in tran sgenic tobacco were bigger
，
and the width of the pore was smaller(Fig．2：A)．But
in the lower epiderm is．they are both bigger than wild
type(Fig．2：B)．
言2O
毳
要。
舍2O
喜
■ W T 口 3 5s：BnDREB1．
● ●，
l6 21 A l 5
— —
5．97 5．91
． -]．
Stomatal
apparatus
length
Stomatal
apparatus
width
Stomata
length
Upper epidemis
Stoinata
width
【
l 5 5： B
9．88 9 02
． I『]．
Stomata
length
Stomata
width
Lower epidemis
Fig·2 The stomata size and degree of opening of
uppe r an d lower epidermis
Compared with wild types，the stoma densities in
upper an d lower epiderm is of transgenic tobacco were
lower．But the stomata density in upper epiderm is was
different obviously，that of wild—type leaves was 1
． 5
times as much as transgenic plants(Table 1)．
Table 1 Stoma density of leaves between
2．3 The contents of chlorophyll
The contents of chlorophyll in wild—type leaves
were 29％ more than that of transgenic tobacco(Ta—
ble．2)．
= ～
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224 武 汉 植 物 学 研 究 第 25卷
Table 2 The contents of chlorophyll of leaves of
transgenic tobacco and、ⅣT
Basma Chlorophyl content(mS／S)
35S：BnDREB1-5
WT
2．09
2．70
2．4 The analysis of plasma membrane permeabi-
nty
The relative plasma membrane permeability of
wild—type leaves was 1．4 times as much as that of
transgenic leaves after drought stress for 7 h(Fig．3)．
Th e result showed that the ability of transgenie tobacco
drought tolerance was obviously strengthened．
Control Drought
Fig·3 Relative plasma membrane permeability of
lea ves oftransgenic tobacco and wild-type
2·5 Growth and development of transgenic toba．
eeo
Table 3 showed the growth an d development of
transgenic tobacco an d wild—type after being transplan —
ced f0r 22 days．Th e leaf numb er of transgenic tobacco
was more than that of wild·type
． the stalk length and
the height was shorter than that ofwild—type．These da—
ta showed that the growth of transgenic tobacco plants
had been limited．
—
Table 3
—
Growth and development oftransgenic and W T
3 Discussion
Many studies of molecular biology have shown that
ab iotic stress—tolerance mediated by DREB—like tran —
scription factors mainly was some substan ces which c8J1
improve the osmotic regulation in ceHs[ ，
． Th e studies
on the biological properties and physiological process in
transgenie plan t are not revealed．Th e reports of biolo—
gical properties and physiological process in transgenic
plan t are seldom．Comparing with the wild type tobac．
co，we found that the holding water capacity of leaf was
more powerful in tran sgenic tobacco．Naturally，the size
and opening of stomata in upper and lower epiderm is
were observed because water in leaves was transpired
relating to the opening of stomata．Th e results showed
that the stomata density in transgenic tobacco was less
than that in wild—type tobacco．
Stoma in leaf is an important channel which water
and air pass in an d out．The opening and closure of sto—
mata has a signifcant efect on photosynthesis and tran—
spiration．The numb er，size an d opening degree of sto—
mata have direcfly relation to stoma conductivity．Th e
stoma conductivity influences the amount of CO2 from
environment to photosynthetic tissues so that it positive—
ly relates to photosynthetic rate[引
．
Th e ab sorption and accumulation of po tassium is
the crucial regu lation factor in gu ard cel for stomata
opening．Sucrose maintains the osmotic potential an d
regu late stomata closure in guard cel[9】
． Because the
leaf water loss in vitro was less in transgenic tobacc0
than in wild type，the stomata param eter an d the chlo．
rophyl contents were compared after 7 h of leaf water
loss．In transgenic tobacco the stomata density was
less，the stomatal apparatus was larger and the pore was
smaler but in wild—type it was contrary．The results re—
vealed that the development of stomatal apparatus in
transgenic tobacco was beter than in wild—type ．The
dam age degree in wild—type was more serious than in
transgenic tobacco after 7 h of leaf water lOSS
． which
brought out decrease of sensitivity to regulation of sto—
mata opening an d closure in wild—type．Some researehes
indicate that the stomata density has relationship with
photosyn thesis pathway and with diferent life—type of
plant Whether the metabolism process and the strue．
ture of tissue have chan ged in transgenic tobacco wil
be studied further．
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第3期 刘卫群等：DREB类转录因子介导的烟草抗非生物胁迫特性研究(英)
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