全 文 ：武汉植物学研究 2004，22(1)：39~43
Journal of Wuhan Botanical Research
耐盐突变体小麦后代耐盐稳定性分析研究
王鸣刚 ，陈 亮 ，贾敬芬。
(1．厦门大学细胞生物学与肿瘤细胞工程教育部重点实验室，厦门 361005；
2．西北大学生物技术研究室，西安 710069)
摘 要：以卫星搭载小麦种子为原始材料，利用其幼穗、幼胚诱导的愈伤组织进行耐盐突变体的筛选，对耐盐愈伤
组织再生植株后代进行耐盐稳定性生理生化特性分析。结果表明：(1)耐盐系后代在土壤高盐浓度条件下，游离脯
氨酸含量稳定增加，且高于对照系；(2)耐盐系再生植株后代保持较高的K ／Na 比；(3)与对照相比，种子醇溶蛋
白电泳带谱中的 b2b3b5，b7带为耐盐系所特有 b8带消失；(4)耐盐系再生植株后代可溶蛋白电泳带为 26条，而
对照系为 23条蛋白带。其中98 kD、75 kD、52 kD、49 kD和 32 kD为耐盐系的特有蛋白带。而 38 kD和 35 kD蛋白
带为对照系所特有。
关键词：耐盐系小麦；后代；脯氨酸；K ／Na 比；醇溶蛋白；可溶蛋白
中图分类号 ：Q946 文献标识码：A 文章编号：1000 470X(2004)01 0039—05
Study on Physiological and Biochemical Characterization in Progenies of
Selection of NaC!一tolerant W heat M utant by in vitro
WANG Ming—Gang ，CHEN Liang ，JIA Jing—Fen。
(1．The Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cel Engineering．Xiamen
University，Xiamen 361005，China；2．Biotechnology Institute。Northwest University，Xi’an 710069，China)
Abstract：Salt—tolerant callus was selected from immature embryo and young inflorescence seg—
ments of wheat(Triticum aestivum)which seeds were carried by spacecraft。and the salt tolerant
stability of their progenies were studied on physiological and biochemical characterization．The re—
sults showed that：(1)The relative content of free proline in salt—selected wheat plantlet’S pro—
gencies was higher than that in unselected ones at higher NaC1 concentration soils：(2)The salt—
selected lines had higher K+／Na+ratio；(3)Four additional specific electrophoretic bands of
gliadin (b2，b3，b5 and b7)were detected and one band (b8)disappeared in F-seeds from salt—se—
lected lines；(4)26 electrophoretic bands of soluble protein were observed in F4 plants from salt—
selected lines，whereas only 23 bands in control lines．Of which 98 kD，75 kD，52 kD ，49 kD and
32 kD bands were specific in salt—tolerant line，38 kD and 35 kD protein bands only appeared in
control lines．
Key words：Salt—tolerant wheat；Progenies；Proline；K ／Na+ratio；Gliadin；Soluble protein
Salt·-affected soils is a serious problem in agri．-
culture throughout the world．Considerable efforts
have been made to explore salt．-tolerance mecha．
nism in plants in order to improve crop production
under saline conditions．Although a wide range of
genetic adaptions to saline conditions and a number
Received date：2003—05—27，Accepted date：2003—09—08．
Foundation item：The key program on biotechnology in Gansu Province(GK911—3—23)．
Biography：WANG Ming—Gang(1962一)，male，Ph．D．，research interest maily in cel and molecule biology of plant(E—mail：mgwang@
163．corn)．
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40 武 汉 植 物 学 研 究 第 22卷
of significant physiological responses that associa—
ted with tolerance have been observed，and some
advances obtained[ ’ ．the underlying mechanism of
salt tolerance in plants is still poorly understood
today．Several evidences indicate that the physio—
logical and genetic basis of plants is altered when
they are exposed to salt stress which can result in
an enhanced ability to tolerance stress．Enhancing
salt tolerance of crops by cell engineering has at—
tracted world—wide attention in recent years．But
reports specifically on the underling mechanism of
salt tolerance in wheat were rather feWE3,43．Since
the hydroxyproline resistant line could appeared
saIt tolerance[5。，selection under hydroxyproline
stress was used as the primary step in this work，
followed by selection under NaCl stress．For under—
stand the salt—tolerance mechanism ，our aim of this
work Was to produce NaC1一tolerant mutant of
wheat by salt selected calluses from immature em—
bryo and young inflorescence segments，which ger—
minated from seeds treated with spacecraft carry—
ing in space conditions，in order to obtained more
variant types of wheat and to study"the physiologi—
cal and biochemical characteristics of their regener—
ated plantlet’s progenies．
1 M aterials and M ethods
1．1 Induction of embryogenic callus
Embryogenic calluses of cultivars of wheat
(Triticum aestivum cv．Baofeng 7228)from imma—
ture embryo and young inflorescence segments
which germinated from seeds treated with space—
craft carrying in space conditions were used for se—
lecting NaCI—tolerant calluses．The callus induction
medium was N6+2 mg／L 2，4-D+0．5 mg／L KT+
1 50 mg／L glutamin+2 mg／L glycine+ 500 mg／L
CH+30 g／L sucrose(Abbreviations：2，4-D一2，4一
dichlorophenoxyacetic acid，KT — Kinetin，CH —
Casein hydrolysate，NAA = a—naphthaleneacetic
acid，6BA一 6一benzyladenine)．
1．2 Selection of salt—tolerant callus
To select the salt—tolerant line。the embryo—
genic calluses were placed on the same N 6 medium
with induction of embryogenic callus，supplemen—
ted with 50 mmol／L hydroxyproline，4 weeks later
the survival calluses were selected and subcultured
onto fresh media containing 1．4 NaC1 every 4
weeks．After nine passages，the NaC1一selected line
was identified for which grew well and exhibited no
discoloration in the presence of NaC1．This line was
referred to as salt—tolerant line and the initial callus
cultured in NaCl—free medium were used as con—
tro1．
1．3 Plantlets regeneration and seeds propagation
The MS medium plus 1 mg／L 6BA，0．5 mg／L
NAA and 1．2 NaC1 was used for plantlets rege—
neration．The regeneration was performed under
illumination of 3 000 lx with 1 4 h photoperoid at
(25± 2)℃ ．W hen the seedlings grown about 8—
10 cm ，they were transferred to indoor soil and wa—
tered with 1 NaC1 solution．The grains(F1)from
the regenerated plant were cultured again to propa—
gate F2 seeds，and Fs seeds were obtained．The Fs
seeds were planted in salinized soil in which con—
tains total salts higher than 0．6 to obtain F4
seeds．
1．4 Free proline measurement
Free proline was determined according to the
method of Bate[引．Each 100 mg fresh leaves from
the F4 seedlings that planted in salinized soils con—
taining different NaC1 concerntration (0 ，
0．45％，0．78 ，1．07％，and 1．34 ，respectively)
for two weeks were homogenized in 10 mL of 3
sulphosalicylic acid．The homogenate was cen—
trifuged for 20 min at 4 000 r／min and 2．0 mL su—
pernatant was used for proline quantification．The
mean values were calculated from 3 replicates．
1．5 Na and K ions estimation
1 00 mg fresh leaves of F4 seedlings germinated
in different concentration of NaC1 was digested in
60 perchloric acid，then Na and K ions were de—
termined by means of an atomic absorption spec—
trophotometer(Hitachi 1 80—8O Semans)．It repeat—
ed 3 times，and the mean
1．6 Electrophoresis of
teins
value was calculated．
gliadin and soluble pro—
According to the method of Fu[ ，the single F4
dry grain was milled in microhammer mill because
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第 1期 王鸣刚等：耐盐突变体小麦后代耐盐稳定性分析研究(英) 41
the F4 seeds of selected salt—tolerant line have simi—
lar electrophoresis pattern of gliadin，then trans—
fered it into 1．5 mL Ependoff tube contained
0．1 mL of ethylene glycol，and then the gliadin
were extracted for 12 h at 25℃．The gliadin ex—
tracts were clarified by centrifugating at 1 5 000 r／
min for 10 min at 4℃ ，then 50 L of supernatant
into another Ependoff tube with 10ⅡL 0．01 mol／L
formate and 0．1 0A methyl green for electrophore—
sis．The separating polyacrylamide gel concentra—
tion was 6％ and the stacking gel was 3 ．The up—
per electrode一(anode) buffer was 0．2 formate
and 0．1 0A formate at the lower electrode一(catho-
de)．Electrophoresis was performed at 500 V for
2．5 h．
To analyze for soluble protein in progeny
seedlings，leaves of F4 seedling were homogenized
in the mixture of 0．6 0A Tris and 2．88 0A glycine，
after centrifuged at 15 000 r／min for 10 min at
4。C ，the supernatant were used for SDS—PAGE
analysis according to Laemmli[引．The separating
gel was 15 (w／v)and the stacking gel was 3
(w／v)．Soluble protein contents were obtained ac-
cording to Bradford[ ．150 p．g protein was loaded to
each lane．
2 Results and Discussion
2．1 Analysis of free proline
Under saline stress，the proline accumulation
is a marked and wide phenomenon[ 。。．The relative
content of free proline in the control line increased
gradually with the increase of NaC1 concentration
(Table 1)，but when NaCl concentration up to
0．45％，the control lines grow slowly and die out
after short growth period at NaC1 concentration
1．34 ，and the relative contents of free proline in
control did not been measured．But at 1．34 0A NaCl
the relative contents of free proline in salt—tolerant
line were 1 1．33 fold higher than at that of start—
point zero concentration．
Table 1 Relative contents of free proline in F．seedings
planted jn salinized soils for two week~
Note：The values are mean standard error for three
replicates．
The salt tolerance in whole plants is the final
purpose．However，in some cases the cellular salt
tolerance has not always correlated with whole
plant salt tolerance[ u．In this study，the determina—
tion of proline in F4 seedlings indicated that the
progeny of salt—tolerant line grown in salinized
soils expressed higher proline content (Table 1)
and higher K+／Na+ratio(Table 2)．Although the
opinion was different about proline accumula—
tion[ 引，it has been accepted that proline can act as
osmoprotectant．It has been also reported that the
level of mRNA for P5CS (△ 一pyrroline一5一carboxy—
late synthetase)and the level of proline in salt—to—
lerant rice are higher than that in salt—sensitive rice
under high—salinity conditions[ 引．Kishor et a1．[ 。]
also shown that the overproduction of proline in
transgenic tobacco plants resulted in the cells to
maintain osmotic potential and thus enhanced the
salt—tolerance．From above，it was suggested that
accumulation of proline play an important role in
sah—tolerance in wheat．Our study was correlated
with the opinon that proline is as osmoprotectant．
Table 2 Comparison of the content(Pg／g DW )of Na and K ion in F4 seedings planted
jn salinized soils for two weeks
Note：The values are mean standard error for three replicates．
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42 武 汉 植 物 学 研 究 第 22卷
2．2 Comparison of the content of Na and K ion
in F·seeding
In plant，the osmotic stress and ionic stress are
two components of salt stress．In saline environ．
ment，osmotic adjustment are achieved by synthe—
sizing organic solutes(such as proline)and accu—
mulating ions from external environment[13，1 41
．
Analysis of internal K and Na concentration
(Table 2)showed that Na contents in the saIt—
tolerant and control seeding increased when both of
them were grown in NaCl containing soils． Howe—
ver，Na accumulation was higher in the former
than those in the latter．The K level in the control
and salt—tolerant line decreased with increasing
NaCl concentration in the salinizd，and than it de—
creased much more rapidly in the former than that
in the latter．Therefore the salt—tolerant seedings
maintained relative higher K ／Na ratio．During
NaC1 stress，the plants induce processes that regu—
late intracellular ion accumulation and vacuole
compartmentation and mediate osmotic adjustment
and moderate cytoplasmic ion activities．These data
were corresponded with the ideas from Niu[1S~
． 0ur
results were correlated with their opinon that salt—
tolerant plant can accumulated higher concentra—
tion K ion．
2．3 Changes of gliadin of F．seeds
Glutenin，i．e．the storage protein of wheat an—
dosperm，accounts about 50％ of the total wheat
grain proteins，which could be divided into 40——50
different components．These gliadin were closely
related with the chromosomalloci of genes control—
ling their synthesis．The gliadin constructed the
‘Fingerprint’of wheat variety[as]．The changes of
gliadin reflect their genotype．Therefore it could be
used as a useful marker to distinguish between the
salt—tolerant wheat mutant and their original vari—
eties．The results of PAGE of gliadin revealed that
13 bands of gliadin appearing in NaC1一tolerant seed
line(Fig．1：lane 2)because the F4 seeds of selected
salt—tolerant line have similar electrophoresis pat—
tern of gliadin．The additional bands of gliadin
were b2，b3，b5．b7，respectively and deletional
band was b8 as compared with the control(Fig．1：
lane 1)．The appearance of specific bands of gliadin
might reflect the synthesis change of gliadin of
grain in salt—tolerant line．Although it is not possi
—
ble to identify the specific function to such gliadins
at present’it can be suggested that those gliadins
maybe advantageous to the maturity of the salt—to1
一
erant wheat seeds grown in salinized soils
．
2
一 b2
+一 b3
．。。b5
+一 b7
,~-b8
Lane l：Control seeds；Lane 2；Sah—tolerant seeds．
Arrows indicated the changed bands
Fig·1 The electrophoresis photograp~ of
gliadin in F·seeds
2．4 Analysis of SDS-PAGE of soluble protein of
F．seedlings
Several new proteins which are synthesized in
response to an alternative environment have been
reported as ‘stress protein’in plants[ ．However
only a few of these proteins have been found to be
involved in known physiological or metabolic pro—
cesses．By comparing to the SDS—PAGE map，it can
be found that there are difference in protein pat—
terns between the salt—tolerant line(Fig．2：lane 2)
and the control line (Fig．2：lane 1)．There are
some specific proteins in salt—tolerant line．It re—
vealed that there are 26 protein bands in salt—
tolerant line，and 23 protein bands in contro1．The
98 kD，75 kD，52 kD，49 kD and 32 kD proteins
were specific in salt—tolerant line(Fig．2：lane 2)，
while 38 kD and 35 kD proteins only present in
control(Fig．2-lane 1)．The appearance of specific
protein band might reflect the synthesis of salt
adapted protein．Although it is not possible to as．
sign the specific function to such protein at present
at this work，they can be used to further study on
the regulation of gene expression during exposure
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第 1期 王鸣刚等：耐盐突变体小麦后代耐盐稳定性分析研究(英) 43
to increased levels of NaCl stress．The identifica—
tion and quantitation of these proteins may provide
a correlation between the altered expression of spe—
cific genes and changes in the environment．
3 1 2
+一98 kD
+一 75 kD
52 kD
# 49 kD
#i；船D
+一 32 kD
Lane 1：Salt—tolerant seed；Lane 2：Control seed；
Lane 3：Protein markers．
Arrows indicated the changed bands
Fig．2 The electrophoresis photograph of
soluble protein in F4 seedlings
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