全 文 ：Vol. 30 , No. 12
pp. 1192 - 1198 　Dec. , 2004
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 30 卷 第 12 期
2004 年 12 月 　1192～1198 页
Molecular Cloning , Expression and Activity Assay of Thioredoxin h —A Gene Re2
lated to Self2incompatibility in Leymus chinensis
ZHANG Wei2Dong1 , LIU Gong2She1 , 3 , CHEN Shuang2Yan1 , LI Xiao2Feng1 , LI Hong2Jie1 ,2 Ξ
(1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology , Institute of Botany , Chinese Academy of Sciences , Beijing 100093 , China ;2 Current
address : Department of Plant Pathology , Washington State University , P. O . Box 646430 , Pullman WA 9916426430 , USA)
Abstract 　Leymus chinensis (Trin. ) Tzvel. , which is widely distributed in East Asia , is an important self2incompatible forage species
in Poaceae. h2type thioredoxins are a family of small proteins that exists throughout the animal , plant , and bacterial kingdoms. Gene for
thioredoxin h expressed in mature pollens is related to self2incompatibility in some grasses species. In the present study , DNA and cDNA
sequences of thioredoxin h ( TrxLc) gene were cloned from L . chinensis . The cDNA nucleotide sequence of TrxLc had 53. 5 % -
5614 % of homology to thioredoxin h genes that originated from other self2incompatible grasses. Using Southern blotting , gene TrxLc was
estimated to be one copy in L . chinensis genome. Northern blotting indicated that TrxLc did not express in leaves , roots , stems , and
young pistils , but expressed in young pollens and mature pistils. The highest expression level of TrxLc was detected in mature pollens.
Gene TrxLc was expressed as recombinant protein in Escherichia coli and tested for thioredoxin activity. In the nonspecific insulin reduc2
tion assay , the protein displayed typical thioredoxin activity and had similar values of Vmax and Km with h2type thioredoxins of other plant
species , but was not similar with that of E. coli .
Key words 　TrxLc (thioredoxin h of Leymus chinensis) ; Leymus chinensis ; Self2incompatibility ; Gene cloning ; Enzyme assay
羊草自交不亲和性相关基因 TrxLc 的克隆、表达和酶活性分析
张卫东1 　刘公社1 , 3 　陈双燕1 　李晓峰1 　李洪杰1 ,2
(1 中国科学院植物研究所光合作用和环境分子生理学重点实验室 , 北京 100093 ;2 华盛顿州立大学植物病理学系 , 普尔曼 646430 ,华盛
顿州 ,美国)
摘 　要 　羊草是一种重要的禾本科牧草 ,主要分布于亚洲东部地区。硫氧化还原蛋白 H (Trx h) 是一大类广泛分布于动
物、植物和微生物中的小分子蛋白。在一些禾本科植物中 ,一种在成熟花粉中表达的 Trx h ,与植物的自交不亲和性有
关。本研究克隆了羊草花粉硫氧还蛋白基因 ( TrxLc)的 DNA 和 cDNA 序列 , 与其他禾本科植物 Trx h 的 cDNA 序列相比 ,
它们具有 53. 5 %～5614 %的同源性。Southern 杂交显示 TrxLc 在羊草基因组中是单拷贝。Northern 杂交显示 TrxLc 在羊草
根、茎和叶中没有表达 , 在成熟雌蕊和幼小花粉中微量表达 , 但是在成熟花粉中大量表达。在大肠杆菌中表达重组
TrxLc并检测了其活性。胰岛素还原反应中得到的 Vmax值和 Km 值显示 , TrxLc 类似于其他植物的非花粉组织特异表达的
Trx h ,但与大肠杆菌的 Trx h 差异较大。
关键词 　TrxLc (羊草硫氧化还原蛋白 H) ;羊草 ;自交不亲和 ;基因克隆 ;酶活性
中图分类号 :S512
　　Thioredoxins are an important family of small pro2
teins that function in regulation of many cell events
throughout the animal , plant , and bacterial kingdoms[1 ] .
Thioredoxins undergo reversible redox changes through an
active disulfide center , which in a highly conserved se2
quence [2Trp2Cys2Gly ( Ala )2Pro2Cys2] [1 ] . Comparing with nonphotosynthetic organisms , plants are unique inpossessing three types of thioredoxins that are located indifferent cellular compartments. The f2type and the m2type thioredoxins are located in chloroplasts and involvedin the light regulation of photosynthetic enzymes[2 ,3 ] . Theh2type is believed to be cytosolic. In plants , h2type thi2ΞFoundation item : Sustainable development of grassland in semi2agricultural and semi2pastoral area , Chinese Academy of Sciences (Nk“10. 5”. A205) and
evaluation of germplasm by molecular markers in Leymus chinensis ,Ministry of Science and Technology(2001BA707B02) .
Biography : ZHANG Wei2Dong , male , born in Hebei Province , lecture , graduate student of CAS. 3 Author for correspondence : LIU Gong2She , principle
investigator in Institute of Botany , CAS. Field of research : Plant developmental biology.
Received(收稿日期) :2003212230 ,Accepted (接受日期) : 2004205230.

oredoxins can be reduced chemically by the physiological
regent , NADH 2 thioredoxin reductase (NTR) , and they
could in turn further reduce target proteins , which result
in structural changes and play some physiological roles in
the organisms[4 ] .
NADH + H + + Thioredoxinox ———NADH + + Thioredoxinred
22SH
Target proteinox + Thioredoxinred ———Target proteinred + Thioredoxinox
　　　　　　22SH　　　　　　　　　　　　　　S2S
Target proteinred ———Physiological roles
Four tissue2specific genes for thioredoxin h have
been identified in different plant species. Thioredoxin h
in wheat seeds can improve seed germination by reducing
seed storage proteins and inactivating amylolytic enzy2
mes
[5 ,6 ]
. Thioredoxin h in stigma of Brassica napus was
found to interact with the self2incompatible locus receptor
kinase (SRK) , which is the female component of self2in2
compatible system in genus Brassica [7 ,8 ] . Functions of
thioredoxin h in sieve tubes of rice ( Oryza sativa L. )
have been proposed including oxidative protection , differ2
entiation of vascular tissue , or as a messenger
molecule[9 ,10 ] . Thioredoxin h in mature pollens of Pha2
laris coerulescens might influence its self2incompatibility[11 ]
and Juttner et al . [12 ] cloned a distinct subclass of plant
thioredoxin h from mature pollens of several other grass2
es . However , there is no information on the complete
DNA sequences of thioredoxins h , the copy number in
genome , and the expressional characteristics in other tis2
sues.
Leymus chinensis (Trin. ) Tzvel . is an important for2
age species of Poaceae widely distributed in East Asia ,
including China , Korea , Mongolia , and Eastern
Russia[13 ] . The high production and nutritive value make
the species important for animal husbandry in northeastern
China[14 ] and there is an increasing demand for the grass
to restore and conserve the deteriorating natural
grasslands[15 ] . L . chinensis is also a self2incompatible
forage[16 ] . Whether the species possesses the thioredoxin
h in mature pollen and the chemical characteristics of the
thioredoxin h are not known.
In this paper , we report on cloning of genomic DNA
(gDNA) and cDNA sequences encoding L . chinensis thi2
oredoxin h ( TrxLc) . The copy number of TrxLc in L .
chinensis genome , location of expression , and the
catalytic properties after production and purification of the
active recombinant TrxLc in E. coli were analyzed. The
results presented in this study might be useful for chang2
ing its self2incompatibility and improving the quality of L .
chinensis by gene engineering. The information also has
scientific significance for thioredoxin h evolution in plants.
1 　Materials and Methods
1. 1 　Materials and reagents
　　Plants of Nongmu 1 , a cultivar of Leymus chinensis ,
were grown in greenhouse. Samples of shoots , leaves ,
and roots were obtained from 12week2old plants. Pollens
and pistiles were collected at uninucleate microspore stage
(immature anthers) and trinucleate microspore stage (ma2
ture anthers) . Anthers less than 1. 8 mm in length were
defined as‘uninucleate’, and anthers that were ready to
dehiscence were regarded as‘trinucleate’[17 ] . Tissues
were stored at - 80 ℃until use.
Reagents and enzymes for cloning and recombinant
DNA manipulation were provided by Promega (Madison ,
WI , USA) , Takara ( Dalian , China ) , or GibcoΠBRL
(Crewe , UK) , if not stated otherwise. Primers were syn2
thesized by Sangon ( Shanghai , China) and DNA and
cDNA were sequenced by GeneCore (Shanghai , China) .
Nucleotide and amino acid sequence analyses were per2
formed with DNAMAN software ( version 5. 0 , Lynnon
Biosoft , Quebec , Canada) . Ni2NTA Superflow was pro2
vided by Qiagen (Hilden , Germany) . NADPH and insu2
lin were from Sigma (St . Louis , USA)
All kinds of bacteria and plasmids were maintained
in Key Laboratory of Photosynthetic and Environmental
Molecular Physiology , Institute of Botany , Chinese
Academy of Sciences , Beijing , P. R. China.
1. 2 　DNA isolation and cloning of the full length of
thioredoxin h
　　Genomic DNA of L . chinensis was isolated by CTAB
method from young leaves[18 ] . To clone the full length
DNA of TrxLc , a pair of degenerate primers was designed
according to the cDNA ends of pollen thioredoxin h from
other plants , such as Phalaris coerulescens , Lolium pe2
renne , Hordeum bulbosum and Secale cereale (those se2
quences were found in GenBank) . The sequences of the
3911　12 期 ZHANG Wei2Dong et al . :Molecular Cloning , Expression and Activity Assay of Thioredoxin h —A Gene Related to. . . 　　　

primers were listed as follows : P1 (forward strand) : 5′2
CCTCTTAATTGCCCGCCAGGAGAT23′; P2 ( reverse st2
rand) : 5′2TCAA ( G) CTGCCATCA ( G) CCAAG ( T) AG
CTT23′.
After initial denaturation for 6 min at 94 ℃, DNA
was amplified by 35 cycles at 94 ℃for 1 min , 58 ℃for 1
min , and 72 ℃for 1 min and 30 s , and finally a 7 min
extension at 72 ℃was followed. The PCR products were
separated on 1. 3 % agarose gels. The glass2milk purified
PCR products were cloned into the p GEM2T vector for se2
quencing.
1. 3 　RNA extraction and cDNA cloning of thioredoxin h
Total RNA was extracted from different tissues using
Trizol reagent as described by the producer. The quality
of RNA was evaluated by gel fractionation with a 1. 2 %
denaturing agarose gel[19 ] . For cDNA cloning , 5μg of to2
tal RNA extracted from mature pollens were used for the
first strand cDNA synthesis following the manufacturer’s
instructions. 100ng of cDNA were used for PCR reaction
using the following primers , which were designed based
on the DNA sequence of thioredoxin h , P3 : 5′2GGA
GGATCC ATGGGGGGCTGTGTGGGAAAG23′, and P4 :
5′2GCA GTCGACTCAGCTGCCATCGCCAAGAGCTTG23′.
BamH Ⅰand Sal Ⅰcleaverage sites (underlined) were
located in the two primers , respectively. PCR was per2
formed at 94 ℃for 6 min , followed by 35 cycles of 94 ℃
for 30 s , 45 ℃for 60 s , and 72 ℃for 60 s. An extension
at 72 ℃for 5 min was carried out before maintaining the
amplified products at 4 ℃. Subsequently , purification and
sequencing of PCR products were performed as before.
1. 4 　Southern blotting and Northern blotting analyses
Aliquots of 10 μg of genomic DNA were digested
with appropriate restriction enzymes , including EcoR Ⅰ,
Hind Ⅲ, Nde Ⅰand Sma Ⅰ, separated on a 0. 8 % aga2
rose gel , and transferred onto a Nylon membrane ( Hy2
bond2N + , Amersham , UK) . The membrane was blotted
with [ 32 P ]2labeled TrxLc DNA probe , and was then
washed twice (15 min each) at 65 ℃ in 0. 1 ×SSC (20
×SSC , NaCl 3 molΠL , trisodium citrate 0. 3 molΠL)
containing 0. 1 % SDS.
Total RNAs isolated from leaves , shoots , roots , pis2
tils , immature and mature pollens were separated on form2
aldehydeΠagarose gel , and transferred onto a Nylon mem2
brane ( Hybond2N + , Amersham , UK) , and then blotted
with [ 32 P]2labeled cDNA of TrxLc .
1. 5 　Thioredoxin h extraction , purif ication , and ex2
pression in E. coli
　　After double digestion of RT2PCR products by
BamH Ⅰ and Sal Ⅰ, a 393 bp restriction fragment ,
which contained the entire coding region of TrxLc , was
generated. The amplified ORF (open reading frame) was
ligated into plasmid pET230a and then transformed into
E. coli strain BL21. The saturated culture was diluted
1∶100 ( VΠV) and , when absorbance at 600 nm reached
0. 5 , isopropyl thio2β2D2galactopyranoside ( IPTG) was
added to 0. 5 mmolΠL. Four hours after induction , cells
were then recovered by centrifugation at 4 000 ×g for 5
min. The cell pellet was resuspended in sonication buffer
(50 mmolΠL Naphosphate , pH 7 , 300 mmol NaCl) and
sonicated briefly on ice to lyse the cells. After centrifuga2
tion , the supernatant was collected and mixed gently with a
50 % ( WΠV) slurry of Ni2NTA resin. The proteins were pu2
rified by the high affinity for Ni2NTA resin due to six con2
secutive histidine residues at the N terminus. The expression
and purification of TrxLc were viewed by SDS2PAGE.
1. 6 　Enzymes assays in thioredoxin2catalysed reduc2
tion of insulin
　　Thioredoxin has been shown to catalyze the nonspe2
cific reduction of insulin disulfide by reducing agent
dithothreitol . Recombinant thioredoxin h catalyzed insulin
reduction , which was monitored as a turbidity increase at
650 nm using spectrophotometer[20 ] . The increase in tur2
bidity was associated with precipitation of the insulinβ2
chain. One microliter of reaction mixture contained 14 U
bovine insulin , 100μmol potassium phosphate , pH 7. 1 ,
0. 5 μmol dithiothreitol , 2 μmol EDTA and variable
amounts of thioredoxin h . The activity is expressed as
Δ A650Πmin.
2 　Results
2. 1 　Isolation and sequence analysis of thioredoxin h
DNA and cDNA
　　In order to obtain gDNA sequence of TrxLc , PCR
was carried out with the primers P1 and P2. A 2 2572bp
DNA fragment was amplified , and the cDNA was ampli2
fied with the primers P3 and P4 from total RNA of mature
pollens ( GenBank : AY204511) . Compared with cRNA
sequence , the gDNA sequence included 3 introns and 4
exons (Fig. 1) .
4911 　　　作 　　物 　　学 　　报 30 卷 　

Fig. 1 Structure of genomic DNA of TrxLc ( Thioredoxin h from Leymus chinensis , 2 257 bp total)
　　In the cDNA sequence , a single ORF was identi2
fied . Upstream of the conserved initiation codon six in2
frame stop codons are found. This conformed the position
of initiation coden and showed that the 5′UTR accounted
for more than 64. 5 % of the total length of the mRNA.
The cDNA of TrxLc contained a 396 bp open reading
frame. The TrxLc protein consisted of 131 amino acid res2
idues with a deduced molecular weight of 14 kDa. It had
an active center consensus sequence WCGPC. Compared
with mRNA sequences from P. coerulescens , L . pe2
renne , H. bulbusom , and S . cereale , cDNA sequence
of TrxLc displayed 53. 5 % , 54. 4 % , 55. 6 % , and
5614 % of nucleotide identity. However , the coding re2
gion at the 3′region of the gene displayed high level of
conservation with 94. 2 % , 92. 7 % , 94. 4 % , and
9810 % of nucleotide identity , respectively. The highest
level of variability was found at the 5′region of the gene
with only 3111 % , 33. 3 % , 33. 8 % , and 33. 7 % of nu2
cleotide identity , respectively. Compared with other iden2
tified thioredoxin h existed in wheat , rape , and rice ,
TrxLc showed 30. 86 % , 28. 99 % , and 31. 99 % of iden2
tity in cDNA sequence and 53. 1 % , 50. 5 % , and
5017 % of identity in coding region. Although some nu2
cleotide variations were detected in the coding region ,
only a few of them resulted in amino2acid changes. Gene
TrxLc showed 96. 2 % , 94. 7 % , 96. 2 % , and 9815 % of
amino2acid identity with thioredoxins of P. coerulescens ,
L . perenne , H. bulbosum and S . cereale , respectively.
However , the protein sequence of TrxLc in L . chinensis
exhibited 36. 9 % , 28. 9 % , and 32. 0 % of amino2acid
identity with thioredoxin h protein sequence of wheat ,
rape and rice.
2. 2 　Southern blotting and Northern blotting analyses
The copy number of TrcLc was determined by South2
ern blotting analysis using genomic DNA of L . chinensis
digested with various restriction endonucleases. Digestion
with EcoR Ⅰ, Hind Ⅲ, Nde Ⅰand Sma Ⅰ, which did
not cut within the probe region , resulted in one intensive
hybridization signal in each lane under high stringent con2
dition (Fig. 2) . This indicated that a single copy of gene
TrxLc existed in L . chinensis .
Fig. 2 Southern blot of Leymus chinensis DNA digested
with Eco R Ⅰ, Hind Ⅲ, Nde Ⅰand Sma Ⅰ( Lanes 1 to 4)
using 32 P2labeled TrxLc cDNA as a probe
5911　12 期 ZHANG Wei2Dong et al . :Molecular Cloning , Expression and Activity Assay of Thioredoxin h —A Gene Related to. . . 　　　

The expression of gene TrxLc was detected by North2
ern blotting analysis in various tissues and pollens and
pistils at different developmental stages ( Fig. 3) . Gene
TrxLc expressed in anthers and pistils and no hybridiza2
tion were detected in RNA from leaves , shoots , roots ,
and immature pistils after overexposure to autoradiograms.
Gene TrxLc expressed slightly in immature pollens and
mature pistils , and expressed at the highest level in ma2
ture pollens.
Fig. 3 Expression level of gene TrxLc in Leymus chinensis
Total RNA from the different tissues was electrophoresed on agarose gels , transferred to nylon filters , and hybridized to the TrxLc probe (A)
and 18S rRNA probe as a control (B) ; The signal of TrxLc transcription level were turned into data and showed in column (C) .
2. 3 　Expression and purification of thioredoxin h in
E. coli
　　To express Leymus thioredoxin h in E. coli , the
coding sequence of TrxLc was subcloned in a pET230a ex2
pression with a 6 ×His tag at the N2terminus. SDS2
PAGE analysis of crude extracts from transformed E. coli
showed a high level of a polypeptide with the expected
molecular mass. The expressed protein was purified from
crude extracts by Ni2NTA resin affinity chromatography ,
which yielded highly purified His2tagged thioredoxin h
(Fig. 4) .
2. 4 　Assay of TrxLc activity
The capacity of TrxLc to reduce insulin disulfides
following reduction by dithiothreitol was assessed. The
rate of insulin reduction , monitored by the increase of tur2
bidity , increased as a function of TrxLc concentration
(114 - 318μmolΠL , Fig. 52A) . A Lineweaver2Burk plot
was generated from assay data ( Fig. 52B) . The values of
Vmax (0. 05 Δ A650Πmin) and Km (0. 65 μmolΠL) were
equivalent to those obtained with the h2type thioredoxins
of other plants , such as L . perenne and wheat , but dif2 Fig. 4 SDS2PAGE gel of purifiedthioredoxin h proteinsLane 1 :Control (total cell proteins from uninduced BL21ΠpETrxLc) ; Lane 2 :Unpurified total proteins after IPTGinduction ; Lane 3 :Ni2resin2purified recombinant protein.
6911 　　　作 　　物 　　学 　　报 30 卷 　

ferent from those obtained from E. coli thioredoxin h .
Fig. 5 Dithiothreitol reduction of
insulin catalyzed by TrxLc
(A) For the time2course of insulin reduction with variable amount
of TrxLc. (B) Lineweaver2Burk plot calculated using
different concentrations of TrxLc.
3 　Discussion
In this study , a full2length ( 2 257 bp ) of L .
chinensis thioredoxin h genomic sequence was cloned by
PCR. This is the first report on the full2length thioredoxin
h genomic sequence of mature pollens. The comparison of
cDNA sequences demonstrated that the similarity among
genes for thioredoxin h from mature pollens in different
self2incompatible grasses was higher. However , the simi2
larity of thioredoxin h expressed in different tissues was
lower. These data indicate that thioredoxin h in different
tissues not only has different physiological functions , but
also has different sequence. Sequence analysis indicated
that TrxLc belongs to the same subclass of thioredoxin h
existed in the mature pollens of self2incompatible grasses.
Additionally , thioredoxin h of mature pollens from several
grasses had much greater identity in nucleotide sequence
of coding region and protein sequence , and the 5′end of
cDNA sequence displayed high level of variability. This
shows that the genes expressed in mature pollens are spe2
cies specific.
It has been believed that there exists quite a com2
plexity in the thioredoxin h protein family in plants , such
as Chinese cabbage[21 ] , Arabidopsis thaliana [22 ] , and
tobacco [23 ] . Our Southern analysis under high stringent
conditions demonstrated that L . chinensis contained a
single copy of gene TrxLc .
Gene TrxLc is highly expressed in pollen grains at
the trinucleate microspore stage but not at uninucleate mi2
crospore stage. A high level of TrxLc transcription in tri2
nucleate microspore shows a high metabolic activity and
plays an essential role in the late stage of pollen develop2
ment . According to the classification of genes by Mascare2
nhas[24 ] , TrxLc may be a‘late’anther2specific gene. It
has been reported that most‘late’pollen2expressed genes
produce enzymes involved in the degradation of the middle
lamella region of cell walls to facilitate pollen germination
and pollen tube extension[25 ] . It will be of interest to
identify chemical pathway and the localization of thiore2
doxin h in mature pollens. In order to clarify the active
pathway of TrxLc , further studies are underway to find its
specific substrates according to the strategy proposed by
Yano et al . [6 ]
The catalytic nature of TrxLc is confirmed by the fact
that thioredoxin catalysed reduction occurs faster than in2
sulin reduction by dithiothreitol alone. Thioredoxin h
from L . chinensis is active in the non2specific insulin2re2
duction test and the catalytic parameters of TrxLc are in
good agreement with those from other self2incompatible
grasses , such as L . perenne and P. coerulescens[12 ] . It
has been reported that two thioredoxin h isoforms with
51 % homology in barley seed have different ability to re2
duce insulin , and their temporal and spatial distributions
are also different [26 ] . The high sequence homology and
similar kinetic properties of thioredoxin h in self2incom2
patible grasses suggested genes for thioredoxin h in ma2
ture pollens might have common physiological roles in
self2incompatible grasses.
Thioredoxin h expressed in mature pollens can influ2
ence the self2incompatibility in P. coerulescens [11 ] . Self2
7911　12 期 ZHANG Wei2Dong et al . :Molecular Cloning , Expression and Activity Assay of Thioredoxin h —A Gene Related to. . . 　　　

incompatibility is common in Poaceae , and self2incompat2
ible grasses usually have distinct characteristics such as
lower seed2set . L . chinensis is also self2incompatible and
can be an important Poaceae forage in East Asia. In this
study , we cloned DNA and cDNA sequences of pollen
thioredoxin h from L . chinensis and analyzed its tissue2
expression and enzyme activity. This is useful to under2
stand the gene function and to improve the agricultural
traits of the grass.
Acknowledgments : Our special thanks are due to our co2
workers QI Dong2Mei , CHEN Shuang2Yan , DONG Gui2
Jun and LIU Xiao2Li for their works on this research. The
authors also wish to thank L G Campbell and B A Vick for
critical reading the manuscript .
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