全 文 ：第4 0卷第 2期 上海师范大学学报(自然科学版) Vol ． 40，No． 2
2 0 1 1 年 4 月 Journal of Shanghai Normal University(Natural Sciences) Apr ． ，2 0 1 1
Screening for the regulatory protein factors interacting
with core transcription factor DYT1 for Arabidopsis
tapetum development
SUN Li-cheng，WEI Jia-li，CHU Yan-xia，ZHOU Shu-min，
YUAN Xiao-jun，ZHANG Wei*
(Shanghai Key Laboratory of Bio-energy Crops，Shanghai University，Shanghai 200444，China)
Abstract:DYT1 is a core transcription factor for Arabidopsis tapetum development． Impairment of
DYT1 function causes failure of most tapetum-related functions． However，DYT1 alone is not suffi-
cient for overall tapetum function，suggesting there should be other factors DYT1 works with． Using
DYT1 as bait in yeast two-hybrid system，screening for the protein interacting with DYT1 was per-
formed in this report． 20 positive colonies were obtained． Blast sequence comparison acquired 8 can-
didate sequences，from which 6 candidates were exhibited to encode putative transcription factors or
other transcription-regulating factors． 5 of these regulatory genes were confirmed to be expressed in
anther． Furthermore we found another anther indispensable transcription factor AMS could interact
with DYT1 in yeast，suggesting that these two transcription factors form heterozygous transcription
complex to control the expression of downstream genes in anther development．
Key words:Arabidopsis;tapetum;DYT1;AMS;yeast two-hybrid
CLC number:Q37 Document code:A Article ID:1000-5137(2011)02-0179-07
Received date:2010-11-19
Foundation item:The Science and Technology Commission of Shanghai Municipality (08PJ1405500) ;The National Natural
Science Foundation of China (30870225) ;The Shanghai Municipality Education Commission 2009 Innovation Project of Science
and Technology
Biography:SUN Li-cheng(1986 －) ，male，graduate student，Shanghai Key Laboratory of Bio-energy Crops，Shanghai U-
niversity;ZHANG Wei (1966 －) ，male，professor，Shanghai Key Laboratory of Bio-energy Crops，Shanghai University．
* Corresponding author
1 Introduction
The tapetum is a nutrient tissue of the anther of flowering plants，essential for normal male reproduction．
The impairments of tapetum often lead to male sterility． Up to date，several genes have been identified in the
investigation to the molecular machinery of male sterility of the model plant Arabidopsis thaliana． Among them
DYT1 shows to be prominent since it is so far the earliest expressed gene identified to encode a bHLH tran-
scription factor． dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 5，with excess and
enlarged vacuoles in tapetal cell． Callose dissolution is also deficient in the mutant anther，which occurs at an-
ther stage 8 in wild type tapetum［5］． As early as anther stage 4，DYT1 expression is detected，though with a
weak style，in both the second parietal cell and sporogenous cell［1］． DYT1 expression becomes stronger at the
上海师范大学学报(自然科学版) 2011 年
stage 5，culminates at the late stage 5 and early stage 6，and vanishes at the stage 7． The expression pattern of
DYT1 highly corresponds with the earliest events of the tapetum development，suggesting that DYT1 could be
responsible for most tapetum function，which was further confirmed by the significant down-regulated expres-
sion of most tapetum-specific genes under dyt1 background．
Nevertheless，it has been demonstrated from several sources that DYT1 alone is not sufficient to cause es-
tablishment of intact tapetum normal function． Firstly，ectopic expression of tapetum can not produce signifi-
cant ectopic phenotype［1］． Secondly，the expression levels of some tapetum development genes change slightly
in dyt1 mutant background compared with the wild type，indicating the transcription of these genes are con-
trolled by other regulatory factors． Thus the possibility whether DYT1 works with other regulatory factors togeth-
er to regulate tapetum development is worth deeper investigation． Here，we reported that DYT1 was used as
bait in yeast two-hybrid system;screening for protein interacting with DYT1 was performed． 5 putative tran-
scription factors were found in the screening． Furthermore，the protein encoded by the full-length cDNA of
AMS，another essential factor of tapetum development［4］ was confirmed to form transcription complex with
DYT1 in yeast．
2 Materials and methods
2． 1 Materials
2． 1． 1 Yeast strains，vectors and cDNA library
AH109 and Y187 strains，pGBKT7，pGADT7 vectors were gifts from Dr． Yang Zhongnan lab (Shanghai
Normal University)． Arabidopsis cDNA library was presented by Dr． Ma Hong lab (Fudan University)
2． 1． 2 Plant materials and growth
Wildtype Arabidopsis thaliana Columbia(Col-0)ecotype was used in this research． The plant was grown
under long-day conditions (16 h light /8 h dark)in a － 23℃ greenhouse．
2． 1． 3 Reagents
Restriction enzyme PstI、EcoRI，RT-ACE Reverse transcriptase，PrimerSTAR HS DNA polymerase，DNA
ligation kit were Purchased from Takara company． Peptone、Yeast Extract were purchased from Oxoid Ltd． YP-
DA、SD /-Trp、SD /-Leu、SD /-Leu-Trp、SD /-Leu-Trp-His、SD /-Ade-Leu-Trp-His，X-gal，GAL4 BD antibody
were purchased from Clontech company． Salmon sperm DNA was from BD MatchmakerTM Library Construction
＆ Screening Kits． Horseradish peroxidase conjugated goat anti-mouse antibody IGg were purchased from
Abmart compamy． Chemiluminescent substrate (Prod # 34077)was purchased from Thermo Scientific．
2． 2 Methods
2． 2． 1 Construction of pGBKT7-DYT1 recombinant plasmids and yeast transformation
According to the cDNA sequence of DYT1，the primers for construction of pGBKT7-DYT1 were designed
as follows:DYT1F:5-CC GAATTCatgggtggaggaagcagattt-3;DYT1R:5-GG CCTCGAG ttatggattgcttctcataac-3
(adding the restriction enzyme EcoRI and PstI sites with more 2-3 protective nucleotides at the 5 end offor-
ward and reverse primers，respectively)． The full-length DYT1 coding region was amplified by PCR and cloned
into the pGBKT7． Then the recombinant plasmid was sequenced．
Yeast transformation was performed as described in the ″Yeast Protocols Handbook″［12］．
2． 2． 2 Detection of expressed DYT1-BD fusion protein in yeast
The total protein was extracted from the bait-harboring yeast cell［8］． Boil the sample for 3 min，and load it
onto a SDS polyacrylamide gel． After electrophoresis separation，the protein was assayed by western blot［13］．
The negative control is cell lysis product of untransformed yeast strain AH109．
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第 2 期 孙立成，韦嘉励，褚艳霞，等:筛选与拟南芥绒毡层发育过程中重要的转录因子 DYT1 相互作用的调控蛋白因子
2． 2． 3 Screening the Arabidopsis cDNA library
2． 2． 3． 1 Self activation and toxicity tests of DYT1 bait
Streak pGBKT7-DYT1 /pGADT7 co-transformed AH109 strain on SD /-Trp-Leu and SD /-Trp-Leu-His-Ade
plates respectively． The self-activation was justified by whether the difference of growth activity of co-trans-
formed AH109 between on the two plates is significant． To test the toxicity of the bait，a colony with similar
size of pGBKT7-DYT1 and empty pGBKT7-harboring yeast strains were inoculated into a 3 mL YPDA liquid
medium，respectively． Incubate them at 30℃ with shaking overnight． Then check the OD600 of the cultures to
determine whether pGBKT7-DYT1 is toxic to the host．
2． 2． 3． 2 Two-Hybrid library screening using yeast mating
Yeast mating was performed as described in“MatchmakerTM Gold Yeast Two-Hybrid System”User Manual［8］．
2． 2． 3． 3 Mating efficiency assay
Dilute the mating culture 1000 times and 10000 times，then spread 100μL of each dilute on the following
plates:SD /-Trp、SD /-LeuandSD /-Trp-Leu． After incubating，calculate mating efficiency according to the below
formula:
No． of cfu /mL of diplolds
No． of cfu /mL of limiting partner
× 100 = %Diploids ．
The strain with the lower viability is the“limiting partner”．
2． 2． 4 Confirmation of positive interactions and rescue of the prey plasmid
2． 2． 4． 1 Rescue of the prey plasmid
Inoculate the positive colonies into SD /-Trp-Leu-His-Ade liquid medium respectively，incubating for
3 － 5 d． Transfer the plasmids from yeast strains to E． coli［8］． The prey plasmid-harboring E． coli colonies can
grow on LB /Amp plates and they were used to extract prey plasmids［8］． The extracted plasmids were sent for
sequencing．
2． 2． 4． 2 Confirmation of positive interactions
The positive prey construction /pGBKT7 co-transformed AH109 strains were streaked on the SD /-Trp-Leu
and SD /-Trp-Leu-His-Ade plates to test the self-activation of preys，according to the identical procedure of
self-activation test for the DYT1 bait (2． 2． 4． 1)．
2． 2． 5 Blast analysis of interaction positive candidates
The candidate plasmids which were confirmed free of self-activation were sent for sequencing． The se-
quences of these candidates were committed to blast analysis referring to these websites:http:/ /www． ncbi．
nlm． nih． gov，http:/ /www． arabidopsis． org．
2． 2． 6 Analysis expression patterns of the positive candidates
The candidate genes were committed to expression analysis on the website:http:/ / jsp． weigelworld． org /
expviz / ．
3 Results
3． 1 The construction，identification and expression of recombinant vector pGBKT7-DYT1
To construct the recombinant vector pGBKT7-DYT1，the full-length cDNA fragment of DYT1 gene was
obtained by RT-PCR，and then be integrated into pGBKT7 as described in the Materials and Methods section．
The restriction enzyme digestion identification was illustrated in Figure 1． Double digestion using EcoRI and
PstI produced a 624 bp target band． Sequencing result confirms all sequence of DYT1 cDNA fragment is correct
for further experiment．
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上海师范大学学报(自然科学版) 2011 年
The confirmed pGBKT7-DYT1 vector was transformed into the yeast host AH109［12］． To ensure the con-
struct is transformed successfully，western blot assay was performed． Antibody against GAL4 BD domain was
used to detect the DYT1-BD fusion protein in the host cell． As illustrated in Figure 2 an approximate 45KDa
BD-fused protein band was detected in the transformed AH109 cell，consistent with the DYT1-BD molecular
weight prediction，while no band visible in the untransformed control lane，confirming that pGBK7-DYT1 was
successfully transformed into the host cell and the fusion protein is expressed correctly．
M:DNA marker;2:pGBKT7-DYT1 vector digested
by EcoRⅠand PstⅠ
Figure 1 Construction of pGBKT7-DYT1 recombinant plasmids
and restriction analysis
Lane 1:protein extracted from transformed yeast，The target band of
DYT1-BDfusion indicated by arrow;Lane 2:protein extracted from
untransformed yeast
Figure 2 Western blot analysis of expressed DYT1-BD fusion
protein in yeast
Finally，toxicity assay using pGBKT7-DYT1 and empty pGBKT7 transformed AH109 was done． These
strains were inoculated in 3 mL YPDA liquid medium and incubated overnight． The absorbance values of the
medium liquid of overnight culture are comparable 1． 18 and 1． 20，respectively，meaning no significant differ-
ence in growth，confirming that DYT1-BD impacts less significant toxicity to the host．
3． 2 Screening for protein interacting with DYT1-BD in Arabidopsis cDNA library
Some baits derived from transcription factor fusion often exhibit self activation in the two-hybrid system，
interfering with the screening reliability severely． To estimate the self activation of DYT1-BD，pGBKT7-DYT1
and pGADT7 co-transformed AH109 strain was inoculated in SD /-Trp-Leu and SD /-Trp-Leu-His-Ade plates
respectively． In a result，the co-transformed AH109 shows normal growth on the SD /-Trp-Leu plate，but no
growth on the SD /-Trp-Leu-His-Ade plate，indicating that DYT1 fusion protein exhibits no self activation．
In our experiment，mating is the major strategy for the library screening． The mating efficiency for an ef-
fective screening should be 2% －5% ． In our case，the number of limiting partner(prey library)was estimated
as 8． 8 × 106 cfu /mL and the number of diploids obtained through mating maneuver was 2． 1 × 105 cfu /mL，ex-
hibiting an acceptable 2． 3% mating efficiency． Totally we recovered 46 positive colonies with SD /-Trp-Leu-
His-Ade plate screening． Among them，26 colonies were verified to be self activation products，leaving 20 non-
self activation colonies for further screening procedure．
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第 2 期 孙立成，韦嘉励，褚艳霞，等:筛选与拟南芥绒毡层发育过程中重要的转录因子 DYT1 相互作用的调控蛋白因子
3． 3 Sequencing and blast of the positive colonies
The non-self activation colonies were rescued on LB /Amp plates，and the vector plasmids were extracted
and sequenced． The obtained sequences were used for the blast assay (http:/ /www． ncbi． nlm． nih． gov) (ht-
tp:/ /www． arabidopsis． org)． As for our special goals (to identify DYT1-associated regulatory proteins) ，the
candidates whose sequences were blasted to encode cellular structure proteins or enzymes were excluded from
the further analysis． According to such criteria，8 candidate sequences were identified to encode regulatory
genes listed in the Table 1． Except the No． 2 and No． 8 candidates，all others seem to encode putative tran-
scription factors，including DYT1． The further investigation of expression pattern of these candidates revealed
that 5 of them were expressed in anther (http:/ / jsp． weigelworld． org /expviz /) ，suggesting DYT1 may form
transcription complexes with these regulatory factors in tapetum regulation．
Table 1 Result of BLAST
No． Gene I． D Description Expression patterns
1 AT1G06170 basic helix-loop-helix (bHLH)family protein floral organ specific
2 AT1G62850 translation release factor global expressing
3 AT3G10910 zinc finger (C3HC4-type RING finger)family protein global expressing
4 AT4G31270 transcription factor global expressing
5 AT4G21330 basic helix-loop-helix (bHLH)family protein anther preferential
6 AT1G74500 basic helix-loop-helix (bHLH)family protein root specific
7 AT2G16910 basic helix-loop-helix (bHLH)family protein anther preferential
8 AT2G22360 DNAJ heat shock family protein global expressing
3． 4 DYT1 and AMS can form heterozygous transcription complex in yeast
DDO:SD /-Trp /-Leu;QDO:SD /-Trp /-Leu /-His /-Ade;
X-gal:SD /-Trp /-Leu /-His /-Ade /X-Gal
Figure 3 Results of DYT1 interaction with AMS in yeast
Since there have been several documents a-
bout transcription complexes which are comprised
of bHLH subunits before our report［10，14］，it is
natural that bHLH transcription factors are major
roles as putative DYT1-associated regulatory pro-
teins． Among them，up to date only AMS has
been identified to be another core factor regula-
ting Arabidopsis tapetum development［4］． To fur-
ther confirm that DYT1 and AMS may form heter-
ozygous transcription complex，we amplified full length ORF of AMS and constructed prey vector with AMS
cDNA integrated into pGADT7． As demonstrated in Figure 3，positive colonies of hybrid appeared on the SD /-
Trp-Leu-His-Ade plate meaning AMS-AD did interact with DYT1-BD fusion． Meanwhile，the colonies exhibi-
ted blue when X-gal was added into the medium，providing more solid evidence of positive interaction between
DYT1 and AMS in yeast．
4 Discussion
By employing yeast two-hybrid system，screening Arabidopsis cDNA library for the regulatory factors
which interact with tapetum-regulating transcription factor DYT1 was performed in this report． At the 1st round
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上海师范大学学报(自然科学版) 2011 年
screening，20 positive colonies had been identified to encode proteins interacting with DYT1． Among these pos-
itive colonies，8 candidates were identified to harbor the cDNA sequences encoding putative regulatory protein
factors and 5 of these candidates were expressed in anther． DYT1 was found in the candidate list meaning that
DYT1 may form homozygous transcription complex in its action of tapetum regulation． To further explore the bi-
ological significance of the identification of the putative DYT1-associated regulatory factors，another core tape-
tum-regulating factor AMS was further tested． The result indicates DYT1 and AMS protein can interact with
each other in yeast． In Arabidopsis，there have been reports about transcription complexes in co-controlling de-
velopmental events． In the floral organ identity control，AP3 and PI play identical roles in the stamen and petal
identity control because ap3 and pi mutant exhibit identical phenotype． Co-overexpression of AP3，PI together
with SEP1 can convert Arabidopsis leaves into stamenoid organs［3］． According to our findings，we believe that
the relationship between DYT1 and AMS mimics somewhat that between AP3 and PI． AMS is expressed in the
tapetum for a prolonged period，commencing from early meiosis (anther stage 6)and continuing through to ap-
optosis of tapetum［4］，demonstrating there is a temporal overlap of DYT1 and AMS expression in tapetum． Mo-
reover，dyt1 and ams exhibit many similar phenotypes such as the over-vacuolated tapetal cytoplasm and al-
most collapsed pollen mother cell upon meiosis． Just like the roles of AP3 and PI in floral organ identity con-
trol，DYT1 and AMS much likely form heterozygous transcription complex to synergistically control tapetum de-
velopment in Arabidopsis．
Acknowledgments
We are very grateful to Yang Zhongnan Laboratory and Ma Hong laboratory for presenting yeast two-hybrid
vectors and Arabidopsis cDNA library． We also appreciate Dr． Zhu Jun in Yang Zhongnan laboratory，Zhu En-
gao in Ma Hong laboratory and Zhang Nan in Song Rentao laboratory for precious technique assistance． This
work is supported by the Science and Technology Commission of Shanghai Municipality (08PJ1405500) ，the
National Natural Science Foundation of China (30870225) ，and the Shanghai Municipality Education Com-
mission 2009 Innovation Project of Science and Technology granted to Zhang Wei．
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第 2 期 孙立成，韦嘉励，褚艳霞，等:筛选与拟南芥绒毡层发育过程中重要的转录因子 DYT1 相互作用的调控蛋白因子
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筛选与拟南芥绒毡层发育过程中重要的转录因子
DYT1 相互作用的调控蛋白因子
孙立成，韦嘉励，褚艳霞，周树敏，袁晓君，张 卫*
(上海大学 上海市能源作物育种及应用重点实验室，上海 200444)
摘 要:DYT1 是拟南芥绒毡层发育过程中重要的转录因子． DYT1 的缺失会导致绒毡层相关功能的缺陷．然而，DYT1 并
不是使绒毡层功能化的充分因素，暗示了 DYT1 蛋白需要和其他转录因子形成复合物才能发挥功能．利用酵母双杂交技
术以 DYT1 蛋白为诱饵在拟南芥 cDNA文库中筛选与 DYT1 相互作用的蛋白．通过筛选从文库中得到 20 个阳性克隆，通
过 BLAST序列比对得到 8 个候选基因序列，从中鉴定出 6 种候选基因编码可能的转录因子或者其他调控转录的蛋白因
子．其中 5 个编码调控因子的基因都被证实在花药中表达．更进一步，发现另一个对花药发育所必需的转录因子 AMS能
与 DYT1 在酵母体内形成转录复合物．表明这 2 个蛋白可能形成异源复合物在花药中共同调节下游基因的表达．
关键词:拟南芥;绒毡层;DYT1;AMS;酵母双杂
(责任编辑:顾浩然)
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