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Overexpression of an ERF transcription factor TSRF1improves rice drought tolerance
Ruidang Quan 1,2,3,Shoujing Hu 1,4,Zhili Zhang 4,Haiwen Zhang 1,2,3,Zhijin Zhang 1,2,3and Rongfeng Huang 1,2,3,*
1Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China 2National Key Facility of Crop Gene Resources and Genetic Improvement,Beijing 100081,China 3National Center for Plant Gene Research (Beijing),Beijing 100081,China 4
Hainan Academy of Agricultural Sciences,Haikou 571000,China
Received 1July 2009;
revised 25November 2009;accepted 30November 2009.
*Correspondence (Tel +861082106143;e-mail rfhuang@https://www.doczj.com/doc/dc4607693.html,)
Keywords:rice,drought tolerance,TSRF1.
Summary
One of the major limitations in rice production is a shortage of water.Conventional breeding as well as emerging genetic engineering methods may be used to improve plant stress tolerance.Some transcription factors regulating stress responsive genes have become important target genes for improving plant drought tolerance.Previ-ously,we have shown that a tomato ethylene response factor (ERF)protein TSRF1that binds to GCC box in the promoters of pathogenesis-related genes positively reg-ulates pathogen resistance in tomato and tobacco,but negatively regulates osmotic response in tobacco.Here,we further report the ability of TSRF1to regulate osmotic and drought responses in monocot rice.TSRF1improves the osmotic and drought tolerance of rice seedlings without growth retardation,as determined by physiologi-cal analyses of root and leaf growth,leaf water loss and survival rate under stress.In addition,the amounts of proline and soluble sugars in transgenic rice lines increase by 30%–60%compared to those in wild-type plants.Moreover,TSRF1activates the expression of a putative rice abscisic acid (ABA)synthesis gene SDR ,resulting in enhanced ABA sensitivity in transgenic rice.TSRF1also increases the expression of MYB,MYC and proline synthesis and photosynthesis-related genes,probably by binding to dehydration responsive element and GCC boxes in promoters of the tar-
get genes.These results demonstrate that TSRF1enhances the osmotic and drought tolerance of rice by modulating the increase in stress responsive gene expression.
Introduction
Rice is a major food for over half of the world’s people,and as populations continue to grow and achieve higher standards of living,the global demand for rice will rise accordingly.As environmental problems worsen,the short-age of available water is becoming more serious,resulting in inadequate water use for rice production,which con-sumes much water.Moreover,drought is one of the most important environmental factors restricting rice yield.Therefore,apart from better water management which requires substantial funds,the use of a biotechnological approach to develop new rice varieties with enhanced
drought tolerance is one of the most ef?cient ways to improve rice production to meet the increasing demand for food.
In addition to traditional breeding,genetic engineering of key stress responsive genes is an effective way to improve plant stress tolerance (Umezawa et al.,2006).Gene targets can be divided into functional genes and reg-ulatory genes (Bhatnagar-Mathur et al.,2008).For instance,the overexpression of functional genes encoding enzymes for the synthesis of osmotic compounds,trans-porters,chaperones and reactive oxygen species scavengers improves drought tolerance in various plants (Umezawa et al.,2006).Furthermore,the application in genetic
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Plant Biotechnology Journal (2010)8,pp.1–13doi:
10.1111/j.1467-7652.2009.00492.x
engineering of transcription factors such as bZIP,NAC,MYB,MYC,zinc-?nger,WRKY and ethylene response fac-tor (ERF)also enhances the drought tolerance of plants as the expression of a given transcription factor can regulate the expression of many stress responsive genes (Bhatnagar-Mathur et al.,2008).In short,the transgenic application of transcription factors may prove to be a powerful approach to modify plant tolerance to abiotic stresses.
Previous reports have shown that ERF proteins,which contain a conserved AP2?ERF DNA-binding domain (Riech-mann et al.,2000),regulate development and responses to environmental stimuli in plants (Nakano et al.,2006).Devel-opmental processes regulated by ERF proteins include embryo development (Boutilier et al.,2002),leaf petiole development (van der Graaff et al.,2000)and leaf epider-mal cell identity (Moose and Sisco,1996),?ower develop-ment (Elliott et al.,1996)and fruit ripening (Wang et al.,2007).ERF proteins also participate in plant responses to biotic stimuli.For example,ERF proteins bind to GCC box (AGCCGCC)and modulate the expression of pathogenesis-related (PR )genes (Ohme-Takagi and Shinshi,1995;Solano et al.,1998;Fujimoto et al.,2000;Gu et al.,2002;Onate-Sanchez and Singh,2002;Onate-Sanchez et al.,2007).Recent investigations (Andriankaja et al.,2007;Middleton et al.,2007)showed that several ERF proteins,such as ERN1,2and 3and EFD from Medicago truncatula ,regulate legume root nodule development to establish a symbiosis with nitrogen-?xing bacteria.
In addition to their involvement in biotic responses,some ERF proteins act as regulators in responses to abiotic stresses including drought,salt and cold.The dehydration responsive element binding proteins (DREB),which belong to the ERF family,were ?rst identi?ed as C-repeat (CRT)?dehydration responsive element (DRE)motif-binding proteins from Arabidopsis (Stockinger et al.,1997;Liu et al.,1998).Overexpression of DREB genes in Arabidopsis activates the expression of many stress-related genes and improves tolerance to drought,salt and cold (Liu et al.,1998;Kasuga et al.,1999;Sakuma et al.,2006).
With the completion of rice genomic sequencing,com-putational analyses revealed that the japonica rice genome has at least 139ERF family genes (Nakano et al.,2006).Moreover,some rice ERF genes are inducible by environ-mental stimuli and are involved in responses to salt,drought,cold and submergence stresses (Dubouzet et al.,2003;Cao et al.,2006;Fukao et al.,2006;Xu et al.,2006;Liu et al.,2007;Hattori et al.,2009).Interestingly,recent investigations of different rice varieties have eluci-dated the escape and quiescence strategies for ?ooding
tolerance.Under ?ooding conditions,ERF proteins SNOR-KEL1and SNORKEL2(SK1and SK2)promote GA accumu-lation and fast stem elongation,known as an escape strategy,in deepwater rice (Hattori et al.,2009),whereas ERF protein SUB1A-1inhibits shoot elongation and enhances survival in submergence-tolerant varieties,known as a quiescence strategy (Xu et al.,2006).In addi-tion,ectopic expression of ERF genes such as HARDY,DREB1A from Arabidopsis ,HvCBF4from barley and TERF1from tomato in rice confers an increased tolerance to abi-otic stresses (Oh et al.,2005,2007;Karaba et al.,2007;Gao et al.,2008).Moreover,the constitutive expression of the rice ERF gene AP37not only enhances the drought tolerance of rice at the vegetative stage,but also increases the grain yield under drought conditions (Oh et al.,2009).Previously,we showed that TSRF1,an ERF protein from tomato,activates the expression of PR genes by binding to GCC box and positively regulates pathogen resistance in tomato and tobacco (Zhang et al.,2004,2007)but nega-tively regulates the osmotic response in tobacco (Zhang et al.,2007).Further studies showed that ABA enhances the binding of TSRF1to GCC box and modi?es the pathogen resistance of tobacco while ?uridone,an inhibitor of ABA biosynthesis,decreases the binding of TSRF1to GCC box (Zhou et al.,2008).These results indicate that TSRF1in?u-ences multiple aspects of plant responses to stress with com-plex cross-reactions.Therefore,in this study,we further examined the role of TSRF1in rice and found the overexpres-sion of TSRF1improves rice osmotic and drought tolerance.
Results
TSRF1and rice ERF proteins have conserved motifs It was reported that at least 139ERF members with a con-served AP2?ERF domain exist in the japonica rice genome (Nakano et al.,2006).To compare the structure of TSRF1with rice ERF proteins,we analysed the sequences of TSRF1and rice ERF proteins using ClustalW and MEME.MEME analysis reveals that TSRF1contains an AP2?ERF domain,a CMIX-1motif,a CMIX-3motif and a CMIX-4motif (Figure 1a),which is similar to an ERF protein At3g23240(ERF1)of group IX in Arabidopsis (Nakano et al.,2006).However,no ERF proteins with all the motifs of TSRF1and At3g23240have been identi?ed in the japonica rice gen-ome by bioinformatics analysis (Nakano et al.,2006).The AP2?ERF domain,containing three b -strands and one a -helix (Allen et al.,1998),is the most conserved motif in TSRF1and the different subgroups of ERF proteins in rice.
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Outside of the AP2?ERF domain,motifs CMIX-1and CMIX-4are conserved in TSRF1and the IXc-subgroup of rice ERF proteins Os07g22770,Os07g22730,Os09g39850and Os03g64260(Figure1b).Interestingly,one putative MAP kinase phosphorylation site,Ser168in TSRF1,is conserved in the IXc-subgroup of rice ERF proteins(Figure1b).In addi-tion,TSRF1also contains a CMIX-3motif which is conserved in the IXa-subgroup of rice ERF proteins Os02g43790, Os01g54890and Os04g46220(Figure1c).
Expression of TSRF1does not affect transgenic rice development
Previously,we found that TSRF1increases pathogen resis-tance in tomato and tobacco plants and decreases osmotic tolerance in tobacco(Zhang et al.,2004,2007).To test whether and how TSRF1functions in rice,we generated TSRF1overexpressing rice lines(Oryza sativa subsp.Japonica cv.Nipponbare)by transformation of constructs containing the coding sequence of TSRF1under the35S promoter.Seven TSRF1overexpressing lines(indicated as OE,Over-xpressor)were con?rmed by RT-PCR ampli?cations (Figure2a),and two of the overexpressor lines were selected for the following assays.
The overexpression of transcription factors often leads to unwanted side effects,such as growth retardation(Liu et al.,1998;Kasuga et al.,1999;Onate-Sanchez et al., 2007;Century et al.,2008).Previously,we did not observe growth retardation of transgenic tobacco plants caused by TSRF1overexpression(Zhang et al.,2007). Therefore,we compared the germination and growth rates of OE lines and wild-type Nipponbare(WT).After 2days of incubation at25°C,the germination rate of the OE lines was not signi?cantly different from that of WT (Figure2b).Further,after7and25days of growth under normal conditions,there was no difference in plant height,root length or fresh weight between the OE lines and WT rice(Figures2–4),indicating that TSRF1overex-pression does not impact the growth and development of transgenic rice under normal growth conditions.
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TSRF1improves rice drought tolerance3
Expression of TSRF1improves osmotic and drought tolerance in rice
The observation that TSRF1overexpression decreases osmotic stress tolerance in tobacco caused us to speculate that unknown factors might suppress the TSRF1-DRE inter-action in tobacco (Zhang et al.,2007).Thus,we investi-gated whether rice have similar factors that affect the TSRF1-DRE interaction and cause osmotic sensitivity in rice.As shown in Figure 3,transgenic lines and wild-type rice seedlings displayed similar growth after incubation in 1?10MS solution for 10days.All leaves of both transgenic and wild-type plants wilted after 3days of treatment with 20%polyethylene glycol (PEG)(Figure 3e–f).However,after a 2-day recovery period in 1?10MS solution,73%–85%of transgenic leaves regained turgor while more than 90%of wild-type leaves remained wilted (Figure 3g).Moreover,3–5new branch roots were generated in the transgenic plants.In contrast,the roots of wild-type plants turned yellow with no new branch root growth (Fig-ure 3f).These results indicate that TSRF1overexpression rice plants recovered faster from osmotic stress than wild-type plants did.We noticed that the OE lines demonstrate similar osmotic tolerance even though the expression level of TSRF1is different among these OE lines (Figure 2a and 3g),re?ecting that a relatively low expression level of tran-scription factors is suf?cient for its function.
Next,we examined the drought tolerance of transgenic rice further.OE and WT rice seedlings exhibited identical growth after 10days in soil (Figure 4a–d).However,upon drought stress elicited by withdrawing water for 6days,more than 90%of WT leaves but <40%of OE leaves curled (Figure 4f).After 2days of rewatering,the survival rate,seedling height,root length and fresh weight were then determined (Figure 4i–m).As shown in Figures 4i and m,85%–94%of TSRF1overexpressing lines survived in contrast to only 23%of wild-type rice.Moreover,the roots of wild-type rice were 34%shorter than those of OE lines,and the fresh weights of whole seedlings and roots of wild-type were 42%and 32%less than those of OE lines,respectively (Figures 4j–l).The earlier mentioned results indicate that TSRF1overexpressing rice is more tol-erant to osmotic and drought stress compared with wild-type.The opposite effects of TSRF1under conditions of osmotic stress in tobacco and rice might be because of different factors in?uencing TSRF1.Further characteriza-tion of these factors will require elucidation of the details of TSRF1regulation in rice and tobacco.
TSRF1increases ABA sensitivity in rice
It was previously reported that TSRF1enhances ABA sensi-tivity during germination,cotyledon expansion and root elongation in tobacco (Zhang et al.,2008).Therefore,we further examined whether TSRF1alters the sensitivity of transgenic rice to exogenous ABA.As shown in Figure 5,without ABA the shoot and root growth were similar in WT and OE lines.However,with ABA treatment,root growth in OE lines was 60%of that in wild-type rice,and the turning green rate of shoots in OE lines was slower than that in wild-type,indicating that TSRF1can enhance rice sensitivity to ABA in a similar manner to the effect in tobacco (Zhang et al.,2008).
TSRF1increases proline and soluble sugar contents in the transgenic rice
Severe osmotic stress causes detrimental changes in cellu-lar components.Accordingly,plants accumulate several metabolites,such as amino acids (e.g.proline),quaternary and other amines (e.g.glycine-betaine and polyamines),and a variety of sugars and sugar alcohols (e.g.mannitol and trehalose)to prevent these detrimental changes (Vino-cur and Altman,2005;Bhatnagar-Mathur et al.,2008).To test whether TSRF1overexpression leads to enhanced osmotic protectant accumulation,we analysed the free
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proline and soluble sugar content in transgenic rice seed-lings at the 2-week stage.The proline content in OE lines was 36%–50%higher than that in wild-type (Figure 6a),and soluble sugars in OE-2and OE-13were higher by 30%and 60%,respectively,compared with those in wild-type seedlings (Figure 6b).The increased osmotic protec-tants in TSRF1overexpression rice plants are bene?cial in protecting plants against drought stress.
TSRF1activates the expression of stress responsive and photosynthesis-related genes in transgenic rice It was previously shown that TSRF1,containing a putative activation domain,is a transcriptional activator (Zhang et al.,2007).The constitutive expression of TSRF1acti-vates the expression of GCC box-containing genes in
transgenic tomato and tobacco (Zhang et al.,2004).Fur-thermore,TSRF1increases ABA content and ABA sensitiv-ity in tobacco (Zhang et al.,2008;Zhou et al.,2008).In this study,we observed that TSRF1also increases the sen-sitivity to ABA in rice.Therefore,we further investigated whether TSRF1affects the expression of ABA synthesis genes in rice.It was reported that short-chain dehydroge-nase ?reductase (SDR )catalyses the oxidation of xanthoxin to abscisic aldehyde,a regulatory step in ABA biosynthesis (Seo and Koshiba,2002).Using the Arabidopsis SDR pro-tein sequence,we searched the rice database and found a putative rice OsSDR gene that might be involved in ABA synthesis in rice.Analysis using qPCR assay indicated that the expression of OsSDR in transgenic OE lines increased threefold compared to that in wild-type seedlings (Figure 7).
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TSRF1improves rice drought tolerance 5
MYB and MYC family transcription factors,both of which respond to drought and ABA in Arabidopsis (Abe et al.,1997,2003)and rice (Dai et al.,2007),are downstream of the ABA-dependent stress response (Shinozaki and Yamag-uchi-Shinozaki,2007).qPCR analysis demonstrated that the expression of OsMYB59and OsMYC1in OE lines dramati-cally increased,compared with that in wild-type (Figure 7).
The increased expression of these regulatory genes might further trigger the expression of downstream target stress responsive genes.Therefore,transcriptome changes induced by constitutive expression of TSRF1will probably enhance the ability of rice to cope with drought stress.
It was reported that photosynthesis,together with cell growth,is among the primary processes affected by
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drought stress(Chaves et al.,2009).Accordingly,the expression of genes related to photosynthesis alters under drought stress in Arabidopsis(Kilian et al.,2007)and rice (Zhou et al.,2007).To determine the effect of TSRF1on the expression of photosynthesis genes,we analysed pho-tosynthesis gene expression in TSRF1overexpressing rice. As shown in Figure7,the expression of OsrbcS,encoding ribulose1,5-bisphosphate carboxylase small subunit,and OsPLAS,encoding plastocyanin,increased in TSRF1over-expressing rice compared with that in wild-type.The expression of the photosynthesis-related genes OsELIP (Os01g0246400,low molecular mass early light-inducible protein HV90),OsPSI-N(Os12g0189400,photosystem I reaction centre subunit N)and OsOEE(Os01g0501800, photosystem II oxygen-evolving complex protein)is inhib-ited by drought stress and induced by48-h rehydration (Zhou et al.,2007).However,we did not?nd signi?cant changes in the expression of these three genes in TSRF1 overexpressing rice.
As TSRF1increased proline accumulation in transgenic rice(Figure6a),we next determined the expression of P5CS1and P5CS2,which encode D1-pyrroline-5-carboxyl-ate synthetase1and2,respectively,catalysing the?rst two steps in proline biosynthesis.As shown in Figure7, the expression of P5CS1and P5CS2increased dramatically in TSRF1transgenic rice,providing the molecular basis upon which TSRF1might transcriptionally modulate the expression of proline biosynthesis.
Promoters of TSRF1-activated genes contain GCC and DRE cis-elements
It was previously shown that TSRF1activates GCC-contain-ing genes in tobacco by binding to GCC and DRE(Zhang et al.,2007).To discover whether promoters of TSRF1-acti-vated genes contain putative cis-elements possibly binding to TSRF1,we searched for cis-elements in the2-kb pro-moter region upstream of ATG in the PLACE database(Higo et al.,1999).As shown in Table1,OsSDR promoter has11 ABRE and1DRE.The possible interaction of TSRF1with OsSDR promoter might contribute to the increased expres-sion of OsSDR,subsequently resulting in ABA accumulation and the ABA-dependent response to drought stress.
In addition,as the downstream transcription factors in the ABA-dependent stress response(Shinozaki and
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Yamaguchi-Shinozaki,2007),rice OsMYB59and OsMYC1promoters contain ABRE,DRE,GCC box and CE1(Table 1).Similarly,the promoter regions of the photosyn-thesis-related genes OsrbcS and OsPLAS contain the cis -elements of ABRE,DRE,GCC box and CE1as well (Table 1).Consistent with the gene expression (Figure 7),we did not ?nd DRE and GCC boxes in the promoter regions of OsPSI-N and OsOEE .Although the promoter of OsELIP has several ABRE and GCC box cis-elements (Table 1),it seems that TSRF1did not activate OsELIP expression (Figure 7).Moreover,P5CS1promoter contains ?ve ABRE,one DRE and four GCC box cis-elements,while P5CS2has one CE1and ?ve ABRE cis -elements (Table 1).In short,as shown earlier,many promoters have several GCC,DRE and CE box elements.Therefore,it is possible that TSRF1might bind to these cis -elements to activate the transcription of these genes because TSRF1is known to bind to GCC,DRE and CE1?GCC in electrophoretic mobility shift assay (EMSA)(Zhang et al.,2004,2007,2008).
Discussion
TSRF1is a candidate gene for drought tolerance engineering in rice
Drought stress triggers a wide variety of plant responses,including changes in gene expression,the accumulation of
osmotically active compounds and the synthesis of speci?c proteins such as hydrophilic proteins and ROS scavenging proteins (Bhatnagar-Mathur et al.,2008).In addition to these functional genes,some regulatory genes have been used in genetic engineering to drought-proof plants.The regulatory proteins involved in stress signal transduction include those affecting protein phosphorylation ?dephos-phorylation,calcium sensing,protein degradation and tran-scriptional control (Bhatnagar-Mathur et al.,2008).Many studies have demonstrated that ERF transcription factors are involved in developmental regulation as well as biotic and abiotic stress responses (Nakano et al.,2006).In the present study,we show that TSRF1overexpression improves osmotic and drought tolerance.And more impor-tantly,TSRF1does not affect the developmental growth of rice under normal growth conditions.It was reported that the constitutive overexpression of rice ERF genes AP37and AP59increased the drought tolerance of rice at the vegeta-tive stage.Interestingly,the overexpression of AP37increased grain yield under severe drought and normal growth conditions,whereas the overexpression of AP59reduced grain yield under both normal and drought condi-tions (Oh et al.,2009).Our preliminary investigations showed no signi?cant difference in grain yield between TSRF1overexpressors and wild-type rice (Data not shown).Therefore,TSRF1might be a candidate gene for genetic engineering in rice to improve drought tolerance.
Table 1cis -elements in promoters of TSRF1-activated genes
Gene ID
cis -elements
OsMYC1Os07g0543000ABRE()1870,)1943),DRE()1422),GCC()1413,)1416,)1425,)1428,)1456,)1830,)2000),CE1?GCC()96,)1393)OsMYB59Os01g0975300ABRE()212),DRE()650),GCC()850,)853,)1469),CE1?GCC ()167,)1214)
OsSDR Os04g0405300ABRE()311,)465,)467,)535,)619,)1125,)1362,)1500,)1547,)1570),DRE()1477),CE1?GCC()968,)1066)OsrbcS Os12g0274700ABRE()744),DRE()715),GCC()319)
OsELIP Os01g0246400ABRE()221,)247,)273,)421,)518,)521),DRE()251),GCC()239,)242,)269,)1538)OsPSI-N Os12g0189400ABRE()16,)234,)240,)1867)OsOEE Os01g0501800ABRE()312)
OsPLAS Os06g0101600ABRE()541,)1126,)1726),DRE()575,)1353,)1564),GCC()519,)1047,)1103),CE1?GCC()952,)1679)OsP5CS1Os05g0455500ABRE()72,)450,)475,)804,)1189),DRE()1122),GCC()299,)503,)591,)1081)OsP5CS2
Os01g0848200
ABRE()818,)1046,)1139,)1331,)1413),CE1?GCC()139)
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Multiple regulatory functions of TSRF1in dicots and monocots
We have previously found that TSRF1transcription is induced by ethylene,salicylic acid in3h,or by the plant pathogen Ralstonia solanacearum after3days of infection. Moreover,the constitutive overexpression of TSRF1in tomato and tobacco activates PR gene expression and enhances resistance to R.solanacearum in transgenic plants (Zhang et al.,2004).Further studies indicated that TSRF1 overexpression enhances broad-spectrum pathogen resis-tance,including resistance to Pseudomonas syringae and Botrytis cinerea,in tomato and tobacco(Zhang et al., 2007).Interestingly,TSRF1expression is induced by manni-tol and NaCl in tomato,whereas TSRF1overexpressing tobacco plants are more sensitive to PEG,mannitol and NaCl(Zhang et al.,2007),suggesting that TSRF1has multi-ple functions in the regulation of responses to pathogens and osmotic stress.In this study,we found that TSRF1acti-vates target genes related to ABA synthesis,MYB and MYC transcription factors,proline synthesis,and photosynthesis, and increases the osmotic and drought tolerance of trans-genic rice.This suggests that TSRF1participates in osmotic response in both tobacco and rice plants with distinct modes of regulation.Currently,we are investigating the effect of TSRF1expression on pathogen resistance in rice. Some ERF proteins interact with other proteins or undergo posttranscriptional modi?cation,both of which might in?uence ERF activity.For example,the interaction of tobacco nitrilase-like protein(NLP)with tobacco ERE-BP2-EREBP3might regulate PR gene expression by seques-tration of EREBPs in the cytoplasm(Xu et al.,1998).The interaction of rice MYC transcription factor OsBP-5with ERF protein OsEBP-89activates waxy gene transcription (Zhu et al.,2003).Moreover,many ERF proteins have sev-eral motifs related to putative phosphorylation sites(Nak-ano et al.,2006),but the phosphorylation of only several ERF proteins has been experimentally veri?ed.For exam-ple,Pto,a Ser?Thr kinase,phosphorylates an ERF protein Pti4,and this modi?cation enhances Pti4binding to the GCC box in vitro(Gu et al.,2000).The phosphorylation of rice OsEREBP1by MAP kinase BWMK1enhances its bind-ing to the GCC box and the transactivation activity of GCC box-mediated transcription(Cheong et al.,2003).In this study,we also found a putative MAP kinase phos-phorylation site,Ser168in TSRF1,suggesting that TSRF1 might undergo posttranslational phosphorylation.
As discussed earlier,ERF proteins interact with other proteins and might be phosphorylated.We propose that TSRF1might interact with other proteins and?or undergo posttranslational modi?cations in plants,both of which in?uence TSRF1DNA-binding activity and transactivation activity.The different interacting proteins or modi?cations of TSRF1in tobacco and rice might explain the distinct TSRF1transactivation activities in tobacco and rice.
Interactions between TSRF1and ABA
ERF proteins regulate responses to biotic and abiotic stres-ses and developmental processes by binding to speci?c cis-acting elements(Alonso and Stepanova,2004).Fur-thermore,ERF proteins regulate the interaction between ethylene and ABA pathways(Pandey et al.,2005;Song et al.,2005;Yang et al.,2005;Wu et al.,2007).For instance,AtERF4overexpression decreases both ABA sensi-tivity to and the expression of ABA-responsive genes in transgenic Arabidopsis(Yang et al.,2005).Similarly, AtERF7overexpression reduces ABA sensitivity of guard cells while AtERF7RNAi increases ABA sensitivity during germination in Arabidopsis(Song et al.,2005).Moreover, ABR1(AP2-like ABA repressor1),a member of the ERF family,is responsive to ABA,and the abr1mutant is hypersensitive to ABA in seed germination and root growth assays(Pandey et al.,2005).These results suggest that the ERF proteins,AtERF4,AtERF7and ABR1,are neg-ative regulators in the ABA pathway.
On the contrary,TSRF1overexpression in tobacco enhances NtSDR expression,increases ABA biosynthesis (Zhou et al.,2008)and increases ABA sensitivity(Zhang et al.,2008).In addition,ABA enhances the interaction of TSRF1with the ABA-responsive element but reduces the interaction of TSRF1with GCC box(Zhang et al.,2008; Zhou et al.,2008),suggesting that TSRF1and ABA regu-late each other and that their interaction is very compli-cated.Similar to the results in tobacco,TSRF1increases OsSDR expression and ABA sensitivity of rice.It remains to be investigated whether ABA modi?es the interaction between TSRF1and its target genes in rice.
Experimental procedures
Plant materials
TSRF1-coding sequence(Zhang et al.,2004)was introduced into rice(Oryza sativa subsp.Japonica cv.Nipponbare)by Agrobacte-rium tumefaciens(Hiei and Komari,2008).The integration of TSRF1into the rice genome was con?rmed by PCR using trans-formed rice genomic DNA as a template.The expression of TSRF1 in transgenic rice plants was further determined by RT-PCR.
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TSRF1improves rice drought tolerance9
Gene expression analysis by qPCR
Total RNA of rice seedlings was extracted with TRIzol reagent.After treatment with DNase to remove genomic DNA contamina-tion,the ?rst strand of cDNA was synthesized by M-MLV reverse transcriptase using 2l g of total RNA as a template.Real-time PCR was performed with a Bio-Rad IQ5system using SYBR to monitor double-stranded DNA products.The PCR primers are listed in Table 2,and Actin was used as an internal control.
Osmotic and drought stress treatment
We determined the osmotic and drought tolerance of TSRF1over-expression rice at T2generation.Rice seeds were incubated at 37°C for 24h and germinated at 25°C under conditions of 16h light ?8h dark for 2days.Then,the germinated seeds were cul-tured with 1?10MS solution for 10days,and rice seedlings were moved to 1?10MS solution with 20%PEG for 3days.Finally,the PEG solution was replaced with a fresh 1?10MS solution for a 2-day recovery.
For drought stress treatment,seeds were germinated as described earlier,and then transferred to soil and grown in a greenhouse at 25°C with 60%–70%relative humidity for 10days.Then,watering was withdrawn for 6days,and rice seedlings were gradually subjected to drought stress.Drought stress-treated rice seedlings were rewatered for 2days,and the survival rate,plant height,root length and fresh weight were determined.
ABA treatment
Rice seeds were germinated at 25°C,with 16h light ?8h dark-ness.At the fourth day of germination,roots had grown to about 0.3cm and seedlings were transferred to 0,1and 5l M ABA for 3days;root length was then determined.
Determination of free proline and soluble sugars in rice leaves
Free proline from rice leaves was extracted with 3%salicylsulfonic acid and determined by colorimetric methods after reaction with ninhydrin (Bates et al.,1973).Soluble sugars in rice leaves were extracted with 80%ethanol and determined spectrophotometri-cally by anthrone reagent using glucose as a standard (Yemm and Willis,1954).
Promoter analysis
Promoter sequences at about 2kb length upstream of the ATG start codon were obtained from Oryzabase (Kurata and Yamazaki,2006),and cis-elements in promoters were searched in the PLACE database (http://www.dna.affrc.go.jp/PLACE/)(Higo et al.,1999).
Sequence analysis of TSRF1and rice orthologs
We performed a multiple alignment analysis with ClustalW2(https://www.doczj.com/doc/dc4607693.html,/Tools/clustalw2/index.html)and printed out the multiple alignment ?les with BOXSHADE 3.21(https://www.doczj.com/doc/dc4607693.html,/software/BOX_form.html).We searched con-served motifs using MEME Version 4.1.1(https://www.doczj.com/doc/dc4607693.html,/meme4_1_1/intro.html)(Bailey and Elkan,1994)and putative MAP kinase sites using GPS Version 2.1(https://www.doczj.com/doc/dc4607693.html,)(Xue et al.,2008).
Acknowledgements
This work was supported by the National Science Founda-tion of China (30730060and 30971699)and National Key Project for Research on Transgenic Biology in China (2008ZX08009-003and 2008ZX001-003).
References
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Table 2Primers used in this article
Gene Accession number Primer sequence Actin NM_001073931F:5￠-ACCTCACCGACTACCTCAT-3￠R:5￠-AGGAAAGAAAGGAGTGGGC-3TSRF1
AAN32899
F:5￠-CGCTGCTCATGAAGAAACA-3￠R:5￠-CAGCACCCAAATCTTCAAAC-3￠
OsMYB59AY569615F:5￠-ATCGCCAAGAGCATTCCTG-3￠R:5￠-AATCCGAGCAGAAGAAGGC-3￠OsMYC1AB110223F:5￠-ACCAGGGAAGACACCCAA-3￠R:5￠-GGTATCAACATCAGAAGGCG-3￠SDR AK110700F:5￠-ACTTTGACCGCGTGCTGG-3￠R:5￠-CGATCGTGACACAAGCCTAAC-3￠Osrbc L22155F:5￠-GAGTTCAGCAAGGTCGGAT-3￠R:5￠-GGGCTTGTAGGCGATGAAG-3￠OsELIP AK062972F:5￠-TAGCAGCAGAAGAACTGAAGAAG-3￠R:5￠-CCGCCATTGCAAATAACTCAAAC-3￠OsPSI-N AK059037F:5￠-GTCGTCTCCAACCAACCAAATTC-3￠R:5￠-ATCCGCACTTGTACTTCTCCTTC-3￠OsOEE AK067715F:5￠-TGCCGAGGAGGCTTACCTTC-3￠R:5￠-CGGTCTCTGGCTTGCTCTTG-3￠OsPLAS AK070447F:5￠-CGCAGACGCAGACCAAGAAG-3￠R:5￠-GCTCGCAGTAGAAGCCGTATG-3￠OsP5CS1D49714F:5￠-TTCTATGGCCTGCACTGTTG-3￠R:5￠-TATATGCATCCACGGCGATA-3￠OsP5CS2
AK101230
F:5￠-CAGATCAAAGCAGCAACCAA-3￠R:5￠-GCTCCTTCACAAGCCACTTC-3￠
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Journal compilationa2010Blackwell Publishing Ltd,Plant Biotechnology Journal,8,1–13
TSRF1improves rice drought tolerance13
１０生物学通报２００５年第４０卷第１１期 ２００３年即Ｗａｔｓｏｎ和Ｃｒｉｃｋ发表ＤＮＡ双螺旋结构５０周年，宣布了人类基因组计划的完成，与此同时，其他许多生物的基因组计划已完成或在进行中，在此过程中产生的大量数据库对科学研究的深远影响是以前任何人未曾预料到的。然而遗憾的是，许多生物学家、化学家和物理学家对这些数据库的使用甚至去何处寻找这些数据库都只有一个比较模糊的概念。 基因转录是遗传信息传递过程中第一个具有高度选择性的环节，近２０年来对基因转录调节的研究一直是基因分子生物学的研究中心和热点，因此亦产生了大量很有价值的数据库资源，对这些数据库的了解将为进一步研究带来极大便利，本文对其中一些数据库进行简要介绍。 １ＤＢＴＳＳ ＤＢＴＳＳ（ＤａｔａＢａｓｅｏｆＴｒａｎｓｃｒｉｐｔｉｏｎａｌＳｔａｒｔＳｉｔｅｓ）由东京大学人类基因组中心维护，网址：ｈｔｔｐ：／／ｄｂｔｓｓ．ｈｇｃ．ｊｐ。最初该数据库收集用实验方法得到的人类基因的ＴＳＳ（ＴｒａｎｓｃｒｉｐｔｉｏｎａｌＳｔａｒｔＳｉｔｅｓ，转录起始位点）数据。对转录起始位点（ＴＳＳ）的确切了解具有非常重要的意义，可更准确的预测翻译起始位点；可用于搜索决定ＴＳＳ的核苷酸序列，而且可更精确地分析上游调控区域（启动子）。自２００２年发布第一版以来已作了多次更新。目前包含的克隆数为１９０９６４个，含盖了１１２３４个基因，在ＳＮＰ数据库中显示了人类基因中的ＳＮＰ位点，而且现在含包含了鼠等其他生物的相关数据。ＤＢＴＳＳ最新的版本为３．０。 在该最新的版本中，还新增了人和鼠可能同源的启动子，目前可以显示３３２４个基因的启动子，通过本地的比对软件ＬＡＬＩＧＮ可以图的形式显示相似的序列元件。另一个新的功能是可进行与已知转录因子结合位点相似的部位的定位，这些存贮在ＴＲＡＮＳＦＡＣ（ｈｔｔｐ：／／ｔｒａｎｓｆａｃ．ｇｂｆ．ｄｅ／ＴＲＡＮＳＦＡＣ／ｉｎｄｅｘ．ｈｔｍｌ）数据库中，免费用于研究，但ＴＲＡＮＳＦＡＣ专业版是商业版本。 ＤＢＴＳＳ对匿名登录的用户是免费的，该网站要求用户在使用前注册，用户注册后即可使用。主页分为２个区域，一个介绍网站的部分信息和用户注册，另一区域为用户操作区，该区约分为１０个部分，可分别进行物种和数据库的选择、ＢＬＡＳＴ、ＳＮＰ以及ＴＦ（转录因子）结合部位搜索等部分。后者的使用可以见网页中的Ｈｅｌｐ部分，里面有比较详细的介绍。ＤＢＴＳＳ还提供了丰富的与其他相关网站的链接，如上文提到的ＴＲＡＮＳＦＡＣ数据库、真核生物启动子数据库（Ｅｕｋａｒｙｏｔ－ｉｃＰｒｏｍｏｔｅｒＤａｔａｂａｓｅ，ｈｔｔｐ：／／ｗｗｗ．ｅｐｄ．ｉｓｂ－ｓｉｂ．ｃｈ／）以及人类和其他生物ｃＤＮＡ全长数据库等。 ２ＪＡＳＰＡＲ ＪＡＳＰＡＲ是有注释的、高质量的多细胞真核生物转录因子结合部位的开放数据库。网址ｈｔｔｐ：／／ｊａｓｐａｒ．ｃｇｂ．ｋｉ．ｓｅ。所有序列均来源于通过实验方法证实能结合转录因子，而且通过严格的筛选，通过筛选后的序列再通过模体（ｍｏｔｉｆ）识别软件ＡＮＮ－Ｓｐｅｃ进行联配。ＡＮＮ－Ｓｐｅｃ利用人工神经网络和吉布斯（Ｇｉｂｂｓ）取样算法寻找特征序列模式。联配后的序列再利用生物学知识进行注释。 目前该数据库收录了１１１个序列模式（ｐｒｏｆｉｌｅｓ），目前仅限于多细胞真核生物。通过主页界面，用户可进行下列操作：１）浏览转录因子（ＴＦ）结合的序列模式；２）通过标识符（ｉｄｅｎｔｉｆｉｅｒ）和注解（ａｎｎｏｔａｔｉｏｎ）搜索序列模式；３）将用户提交的序列模式与数据库中的进行比较；４）利用选定的转录因子搜索特定的核苷酸序列，用户可到ＣｏｎＳｉｔｅ服务器（ｈｔｔｐ：／／ｗｗｗ．ｐｈｙｌｏｆｏｏｔ．ｏｒｇ／ｃｏｎｓｉｔｅ）进行更复杂的查询。ＪＡＳＰＡＲ数据库所有内容可到主页下载。 与相似领域数据库相比，ＪＡＳＰＡＲ具有很明显优势：１）它是一个非冗余可靠的转录因子结合部位序列模式；２）数据的获取不受限制；３）功能强大且有相关的软件工具使用。ＪＡＳＰＡＲ与ＴＲＡＮＳＦＡＣ（一流的ＴＦ数据库）有较明显的差异，后者收录的数据更广泛，但包含不少冗余信息且序列模式的质量参差不齐，是商业数据库，只有一部分是可以免费使用。用户在使用过程中会发现二者的差异，这主要是由于二者对数据的收集是相互独立的。另外该数据库还提供了相关的链接：如ＭａｔＩｎｓｐｅｃｔｏｒ检测转录因子结合部位，网址ｈｔｔｐ：／／ｔｒａｎｓｆａｃ．ｇｂｆ．ｄｅ／ｐｒｏｇｒａｍｓ／ｍａｔｉｎｓｐｅｃｔｏｒ／；ＴＥＳＳ转录元件搜索系统，网址ｈｔｔｐ：／／ｗｗｗ．ｃｂｉｌ．ｕｐｅｎｎ．ｅｄｕ／ｔｅｓｓ／。 转录调节位点和转录因子数据库介绍! 张光亚!!方柏山 （华侨大学生物工程与技术系福建泉州３６２０２１） 摘要转录水平的调控是基因表达最重要的调控水平之一，对转录调节位点和转录因子的研究具有重要意义。介绍了ＤＢＴＳＳ、ＪＡＳＰＡＲ、ＰＲＯＤＯＲＩＣ和ＴＲＲＤ等相关数据库及其特征、内容和使用。 关键词转录调节位点转录因子数据库生物信息学 !基金项目：国务院侨办科研基金资助项目（０５ＱＺＲ０６） !!通讯作者
与
科研探索知识创新与为小波 多分辨率分解尺度，即低通滤波器系数； 分别与低通滤波系数lo-d 、高通滤波系 数Hi-d 各自相乘、累加后，再分别进行下抽取得到低频序列 2淀粉酶启动子区域的W-box 序列结合，从而控制种子糊粉层细胞糖代谢途径的建立。Sun 等（2003）从大麦中分离并纯化出WRKY 型转录因子SUSIBA2，研究表明SUSIBA2是淀粉合成中一个重要的转录调节因子，WRKY 转录因子因此有可能参与碳水化合物的合成代谢。5展望植物WRKY 基因作为植物特有的响应逆境胁迫信号以及调控逆境相关基因表达的重要因子，在植物适应和抵抗逆境过程中具有重要作用。目前对于WRKY 转录因子的研究主要集中WRKY 转录因子在逆境胁迫下的表达模式，从而提高植物对抗各种逆境的能力。但是人们对WRKY 转录因子所介导的植物应答反应及基因调控体系尚不清楚，WRKY 转录因子在各种抗逆信号转导途径及激素信号途径中的信号网络也有待进一步了解。随着分子生物学各种新技术的发展，WRKY 基因在植物抗逆过程中的作用机制将得到更为深入的研究。 参考文献： [1]李蕾,谢丙炎等.WRKY 转录因子及其在植物防御反应中的 作用[J ].分子植物育种,2005,3（3）：401-408. [2]仇玉萍,荆邵娟,付坚等.13个水稻WRKY 基因的克隆及其 表达谱分析[J ]．科学通报，2004，49(18)：1860-1869. [3]3Birnbaum K,Shasha DE,Wang JY ,et al.A gene expression map of the Arabidopsis root [J ].Science,2003,302:1956-1960. [4]4Yoda H et al.Mol Genet Genomics, 2002, 267: 154-161.
植物转录因子及转录调控数据与分析平台 PlantTFDB：植物转录因子数据库 URL: https://www.doczj.com/doc/dc4607693.html, 包含资源：植物转录因子的家族分类规则、基因组转录因子全谱、丰富的注释、转录因子结合图谱(binding motifs)、转录因子预测、系统发生树等 涉及物种：包含拟南芥、水稻、杨树、大豆、玉米、小麦等165个物种。 PlantRegMap：植物转录调控数据与分析平台 URL: https://www.doczj.com/doc/dc4607693.html, 包含资源：植物转录调控元件、植物转录调控网络、转录因子结合位点预测、转录调控预测与富集分析、GO富集分析、上游调控因子富集分析等。 涉及物种：包含拟南芥、水稻、杨树、大豆、玉米、小麦等156个物种。 ATRM: 拟南芥转录调控网络及其结构和演化分析 URL: https://www.doczj.com/doc/dc4607693.html, 包含资源：基于文本挖掘和人工校验的拟南芥转录调控网络、植物转录调控网络的结构和演化特征 涉及物种：拟南芥 植物转录因子及转录调控数据与分析平台(导航页) 我们致力于为广大科研人员提供一个关于植物转录因子和转录调控、集数据和分析于一体的高质量平台，为研究和理解植物转录调控系统保驾护航。 植物转录因子数据库(PlantTFDB) 一套完整的植物转录因子分类规则 覆盖绿色植物各大分支的转录因子全谱 丰富的功能和演化注释 基因组范围的高质量转录因子结合矩阵（156个物种） 在线转录因子预测平台 植物转录调控数据与分析平台(PlantRegMap) 基于高通量实验(ChIP-seq和DNase-seq)和比较基因组方法鉴定的多种转录调控元件 基于转录因子结合矩阵和转录调控元件推测的转录调控网络 涉及165物种的GO注释 一套植物转录调控预测与分析工具，包括转录因子结合位点预测、转录调控预测与富集分析、GO富集分析及上游调控因子富集分析等 拟南芥转录调控网络及其结构和演化特征(ATRM) 基于文本挖掘和人工校验的拟南芥转录调控网络 植物转录调控网络的结构和演化特征
ChIP-Seq技术在转录因子结合位点分析的应用 摘要：染色质免疫沉淀(Chromatin immunoprecipitaion, ChIP)技术是用来研究细胞 内特定基因组区域特定位点与结合蛋白相互作用的技术。将ChIP与第二代高通量测序技术相结合的染色质免疫沉淀测序(chromatin immunoprecipitation followed by sequencing，ChIP-Seq)技术能在短时间内获得大量研究数据，高效地在全基因组范围内检测与组蛋白、转录因子等相互作用的DNA区段，在细胞的基因表达调控网络研究中发挥重要作用。本文 简要介绍了ChIP-Seq技术的基本原理、实验设计和后续数据分析，以及ChIP-Seq技术在 研究转录因子结合位点中的。 关键词：ChIP-Seq；转录因子； 引言 染色质是真核生物基因组DNA主要存在形式，为了阐明真核生物基因表达调控机制，对于蛋白质与DNA在染色质环境下的相互作用的研究是基本途径。转录因子是参与基因表达调控的一类重要的细胞核蛋白质，基因的转录调控是生物基因表达调控层次中最关键的一层，转录因子通过特异性结合调控区域的DNA序列来调控基因转录过程。转录因子由基础转录因子和调控性转录因子两类组成，其中基础转录因子在转录起始位点附近的启动子区，与RNA聚合酶相互作用实现基因的转录；而调控性转录因子一般与位置多样的增强子序列结合，再通过形成增强体在组织发育、细胞分化等基因表达水平调控中发挥极其重要的作用[1]。 ChIP-Seq是近年来新兴的将ChIP与新一代测序技术相结合，在全基因s组范围内分析转录因子结合位点(transcription factor binding sites，TFBS)、组蛋白修饰(histone modification)、核小体定位(nucleosome positioning)和DNA 甲基化(DNA methylation)的高通量方法[2-4]。其中ChIP是全基因组范围内识别DNA与蛋白质体内相互作用的标准方法[5]，最初用于组蛋白修饰研究[6]，后来用于转录因子[7]。同时，新一代测序技术的迅猛发展也将基因组学水平的研究带入了一个新的阶段，使得许多基于全基因组的研究成为可能。相对于传统的基于芯片的ChIP-chip (chromatin immunoprecipitation combined with DNA tiling arrays)，ChIP-seq 提供了一种高分辨率、低噪音、高覆盖率的研究蛋白质-DNA 相互作用的手段[8]，可以应用到任何基因组序列已知的物种，可以研究任何一种DNA 相关蛋白与其靶定DNA 之间的相互作用，并能确切得到每一个片段的序列信息．随着测序成本的降低，ChIP-seq 逐步成为研究基因调控和表观遗传机制的一种常用手段。此外，为了达到更好的检测效果和更为完整的信息，近年来，将ChIP-Seq和ChIP-chip两者融合的研究具有很好的应用前景[9,10]。 转录因子在器官发生过程中起至关重要的作用，在全基因组水平将转录因子定位于靶基因DNA是认识转录调控网络的有效方法之一，了解基因转录调控的关键是识别蛋白质与DNA的相互作用。ChIP-Seq技术能够揭示转录因子的结合位点和确定直接的靶基因序列，可在体内分析特定启动子的分子调控机制，因此被广泛应用于转录调控机制的研究。本文主要就这一技术在转录因子结合位点研究中的基本原理、实验设计和数据分析等技术层面、以及实际应用层面进行讨论。 1 ChIP-seq基本原理及实验设计 1.1 ChIP技术 蛋白质与DNA相互识别是基因转录调控的关键，也是启动基因转录的前提。ChIP是在全基因组范围内检测DNA与蛋白质体内相互作用的标准方法[11］，该技术由Orlando等[12］于1997年创立，最初用于组蛋白修饰的研究，后来广泛应用到转录因子作用位点的研究中[13］。ChIP的基本原理为：活细胞采用甲醛交联后裂解，染色体分离成为一定大小的片段，然后用特异性抗体免疫沉淀目标蛋白与DNA交联的复合物，对特定靶蛋白与DNA片段进行
转录因子 ? 1 简介 ? 2 方法 ? 3 转录因子 转录因子-简介 基因转录有正调控和负调控之分。如细菌基因的负调控机制是当一种阻遏蛋白(repressor protein)结合在受调控的基因上时，基因不表达；而从靶基因上去除阻遏蛋白后，RNA聚合酶识别受调控基因的启动子，使基因得以表达，这是正调控。这种阻遏蛋白是反式作用因子。 转录因子(transcription factor)是起正调控作用的反式作用因子。转录因子是转录起始过程中RNA聚合酶所需的辅助因子。真核生物基因在无转录因子时处于不表达状态，RNA聚合酶自身无法启动基因转录，只有当转录因子(蛋白质)结合在其识别的DNA序列上后，基因才开始表达。 转录因子的结合位点（transcription factor binding site，TFBS）是转录因子调节基因表达时，与mRNA结合的区域。按照常识，转录因子（transcription factor，TF）的结合位点一般应该分布在基因的前端，但是，新的研究发现，人21和22号染色体上，只有22％的转录因子结合位点分布在蛋白编码基因的5'端。 转录因子-方法 这篇文章的试验方法是，通过高密度的寡核苷酸芯片，反映出人21和22号染色体的几乎所有的非重复序列，通过这种芯片，检测三种转录因子，Sp1、 cMyc、和p53的结合位点。结果表明，每种转录因子都有大量的TFBS与之结合。然而，只有22％的转录因子结合位点分布在蛋白编码基因的5'端， 36％的TFBS分布在蛋白编码基因的中部或3'端，并且这36％的TFBS常常和基因组中的非蛋白编码RNA分布在一起。这暗示，在人的基因组中，不仅包含蛋白编码基因，也包含数量相当的非编码基因（noncoding genes），他们都受常见的转录因子所调控。 真核生物在转录时往往需要多种蛋白质因子的协助。一种蛋白质是不是转录机构的一部分往往是通过体外系统看它是否是转录起始所必须的。一般可将这些转录所需的蛋白质分为三大类： (1)RNA聚合酶的亚基，它们是转录必须的，但并不对某一启动子有特异性。 (2)某些转录因子能与RNA聚合酶结合形成起始复合物，但不组成游离聚合酶的
转录因子 基因转录有正调控和负调控之分。如细菌基因的负调控机制是当一种阻遏蛋白(repressor protein)结合在受调控的基因上时，基因不表达；而从靶基因上去除阻遏蛋白后，RNA聚合酶识别受调控基因的启动子，使基因得以表达，这是正调控。这种阻遏蛋白是反式作用因子。而顺式作用因子则指的是基因上与反式作用因子结合的对基因表达起调控作用的基因序列。 转录因子(transcription factor)是起正调控作用的反式作用因子。转录因子是转录起始过程中RNA聚合酶所需的辅助因子。真核生物基因在无转录因子时处于不表达状态，RNA聚合酶自身无法启动基因转录，只有当转录因子(蛋白质)结合在其识别的DNA序列上后，基因才开始表达。 转录因子的结合位点（transcription factor binding site，TFBS）是转录因子调节基因表达时，与mRNA结合的区域。按照常识，转录因子（transcription factor，TF）的结合位点一般应该分布在基因的前端，但是，新的研究发现，人21和22号染色体上，只有22％的转录因子结合位点分布在蛋白编码基因的5'端。 真核生物在转录时往往需要多种蛋白质因子的协助。一种蛋白质是不是转录机构的一部分往往是通过体外系统看它是否是转录起始所必须的。一般可将这些转录所需的蛋白质分为三大类： (1)RNA聚合酶的亚基，它们是转录必须的，但并不对某一启动子有特异性。 (2)某些转录因子能与RNA聚合酶结合形成起始复合物，但不组成游离聚合酶的成分。这些因子可能是所有启动子起始转录所必须的。但亦可能仅是譬如说转录终止所必须的。但是，在这一类因子中，要严格区分开哪些是R NA聚合酶的亚基，哪些仅是辅助因子，是很困难的。 (3)某些转录因子仅与其靶启动子中的特异顺序结合。如果这些顺序存在于启动子中，则这些顺序因子是一般转录机构的一部分。如果这些顺序仅存在于某些种类的启动子中，则识别这些顺序的因子也只是在这些特异启动子上起始转录必须的。 黑腹果蝇的RNA聚合酶需要至少两个转录因子方能在体外起始转录。其中一个是B因子，它与含TATA盒的部位结合。人的因子TFⅡD亦和类似的部位结合。同样，CTF(CAAT结合因子)则与腺病毒的主要晚期启动子中与CAAT盒同源的部位相结合。结合在上游区的另一个转录因子是USF(亦称MLTF)，则可以识别腺病毒晚期启动子中靠近-55的顺序。转录因子Sp1则能和GC盒相结合。在SC40启动子中有多个GC盒，位于-70到-110之间。它们均能和Sp1相结合。然而含有GC盒的不同的DNA顺序与Sp1的亲和力却各不相同。可见GC盒两侧的顺序对Sp1-GC盒的结合究竟如何能影响转录。有时候需要几个转录因子才能起始转录。例如胞苷激酶的启动子需要S p1与GC盒结合和CTF与CAAT盒结合;腺病毒晚期启动子需要TFⅡD与TATA盒结合和USF与其邻近部位相结合。以上所述的因子是一般转录都需要的，似乎并没有什么调节功能。另一些转录因子则可以调控一组特殊基因的转录。热休克基因就是一个很好的例子。真核生物的热休克基因在转录起始点的上游15bp处有一个共同顺序。H STF因子仅在热休克细胞中有活性。它与包括热休克共同顺序在内的一段DNA相结合，所以这个因子的激活可以引起约包括20个基因的一组基因起始转录。在这里，转录因子和RNA聚合酶Ⅱ之间关系很类似细菌的σ因子与核心酶之间的关系。 转录因子是一种具有特殊结构、行使调控基因表达功能的蛋白质分子，也称为反式作用因子。植物中的转录因子分为二种，一种是非特异性转录因子，它们非选择性地调控基因的转录表达，如大麦(Hordeum vulgare) 中的HvCBF2 (C-repeat/DRE binding factor 2) (Xue et al., 2003)。还有一种称为特异型转录因子，它们能够选择性调控某种或某些基因的转录表达。典型的转录因子含有DNA结合区(DNA-binding domain)、转录调控区(acti vation domain)、寡聚化位点(oligomerization site) 以及核定位信号(nuclear localization signal) 等功能区域。这些功能区域决定转录因子的功能和特性(Liu et al., 1999)。DNA结合区带共性的结构主要有：1）HTH 和HL H 结构：由两段α-螺旋夹一段β-折叠构成，α-螺旋与β-折叠之间通过β-转角或成环连接，即螺旋-转角-螺旋结构和螺旋-环-螺旋结构。2）锌指结构：多见于TFIII A 和类固醇激素受体中，由一段富含半胱氨酸的多肽链构成。每四个半光氨酸残基或组氨酸残基螯合一分子Zn2+ ，其余约12-13 个残基则呈指样突出，刚好能嵌入DNA 双螺旋的大沟中而与之相结合。3）亮氨酸拉链结构：多见于真核生物DNA 结合蛋白的 C 端，与癌基因表达调控有关。由两段α - 螺旋平行排列构成，其α - 螺旋中存在每隔7 个残基规律性排列的亮氨酸残基，亮氨酸侧链交替排列而呈拉链状，两条肽链呈钳状与DNA 相结合。
转录因子包括什么主要的功能结构域？其主要的结构特点与功能是什么？ 作为蛋白质的转录因子从功能上分析其结构可包含有不同区域：①DNA结合域（DNA binding domain），多由60-100个氨基酸残基组成的几个亚区组成；②转录激活域（activating domain），常由30-100氨基酸残基组成，这结构域有富含酸性氨基酸、富含谷氨酰胺、富含脯氨酸等不同种类，一酸性结构域最多见； ③连接区，即连接上两个结构域的部分。不与DNA直接结合的转录因子没有DNA 结合域，但能通过转录激活域直接或间接作用与转录复合体而影响转录效率。 与DNA结合的转录因子大多以二聚体形式起作用，与DNA结合的功能域常见有以几种： ①螺旋-转角-螺旋（helix-turn-helix，HTH）及螺旋-环-螺旋(helix-loop-helix，HLH） 这类结构至少有两个α螺旋其间由短肽段形成的转角或环连接，两个这样的motif结构以二聚体形式相连，距离正好相当于DNA一个螺距(3.4nm）,两个α螺旋刚好分别嵌入DNA的深沟。 ②锌指（zinc finger）其结构如图所示，每个重复的“指”状结构约含23个氨基酸残基，锌以4个配价键与4个半胱氨酸、或2个半胱氨酸和2个组氨酸相结合。整个蛋白质分子可有2-9个这样的锌指重复单位。每一个单位可以其指部伸入DNA双螺旋的深沟，接触5个核苷酸。例如与GC盒结合的转录因子SP1 中就有连续的3个锌指重复结构。 ③碱性-亮氨酸拉链（basic leucine zipper，bZIP）这结构的特点是蛋白质分子的肽链上每隔6个氨基酸就有一个亮氨酸残基，结果就导致这些亮氨酸残基都在α螺旋的同一个方向出现。两个相同的结构的两排亮氨酸残基就能以疏水键结合成二聚体，这二聚体的另一端的肽段富含碱性氨基酸残基，借其正电荷与DNA 双螺旋链上带负电荷的磷酸基团结合。若不形成二聚体则对DNA的亲和结合力明显降低。在肝脏、小肠上皮、脂肪细胞和某些脑细胞中有称为C/EBP家族的一大类蛋白质能够与CAAT盒和病毒增强子结合，其特征就是能形成bZIP二聚体结构。
目的：NFAT （Nuclear factor of activated T-cell ）家族在人体生理和病理过程中发挥着重要的作用，但是对NFAT3的转录调节因子却知之甚少。通过本室前期的实验，我们了解到NFAT3可以调节ER （Estrogen Receptors ）的转录活性，因此，反过来，我们检测了ER 对NFAT3转录活性的影响。 方法：通过活性实验、点突变实验及RNA 干扰实验，验证ER 、雌激素及NFAT 激活剂PMA+ION 对NFAT3转录活性的影响，并通过免疫共沉淀（Co-immunoprecipitation ，Co-IP ）实验检测ER 与NFAT3的相互作用，通过染色质免疫共沉淀（CHIP ）实验确定ER α能否被募集到IL-2启动子上，并用核质分离实验，揭示ER α在细胞内对NFAT3定位的影响。 结果： 1． 在293T 及MDA-MB-453细胞中，ERs 可以抑制NFAT3的转录活性。 2． IP 实验证实，NFAT3和ERs 在体内存在相互作用，并且该作用不受NFAT 激活剂PMA+ION 的影响。 3． 敲低293T 细胞中的内源NFAT3后，ER α对NFAT-LUC 活性的抑制作用基 本消除，证实ER α对NFAT-LUC 报告基因的抑制需要NFAT3的参与。 4． 在分别敲低MCF-7细胞中的内源性ER α和ER β后，NFAT3的转录活性都大 幅增强。 5． 构建了两个ER α雌激素结合位点突变体，与野生型ER α相比，两个突变体 对NFAT3活性的抑制依然存在，而在加入雌激素后，对NFAT3的进一步抑制被消除，证实雌激素对NFAT3转录活性的抑制依赖ER 的活性。 6． AKT 和MAPK 重现了ER α磷酸化位点突变体ER α（S167A ）和ER α（S118A ） 对NFAT3转录活性的影响。 7． CHIP 实验证实，加入ER α后，NFAT3与其靶基因IL-2启动子的结合增强， 而加入PMA+ION 后，能进一步促进该结合。 8． 通过核质分离实验发现ER α促使NFAT3入核，并且加入PMA+ION 后， NFAT3的表达增强。 结论：ER α是NFAT3的一个转录调控因子，ER α对NFAT3的转录调节受到雌激素和PMA+ION 的影响。对受NFAT3影响的多种免疫疾病、神经性疾病、心血管疾病和癌症的研究和治疗提供了新的线索。 关键词：ER ，NFAT3，相互作用，磷酸化，转录活性 致谢：该工作得到国家自然科学基金（30530320，30625035，30500191），973计划 P2-44 ER 对雌激素共调节因子NFAT3的转录调节 秦玺，王晓辉，杨智洪，丁丽华，徐小洁，程龙，牛畅，孙慧伟，张浩，叶棋浓 军事医学科学院生物工程研究所，北京市海淀区太平路27号院工作区北楼2121，100850 (xiba315@https://www.doczj.com/doc/dc4607693.html,)
关于组蛋白、甲基化、转录因子、结合位点和CHIP-Seq 1)染色质：真核细胞分裂间期的细胞核内的一种物质，这种物质的基本化学成分为脱氧核 糖核酸核蛋白(核蛋白就是由DNA或RNA与蛋白质形成的复合体)，主要由DNA和组蛋白构成，也含有少量的非组蛋白和RNA。由于它可以被碱性的染料染色，所以称为染色质。在细胞的有丝分裂期，染色质经过螺旋、折叠，包装成了染色体。 2)核小体：核小体是染色体的基本结构单位，由DNA和组蛋白(histone)构成，是染色质(染 色体)的基本结构单位。由4种组蛋白H2A、H2B、H3和H4，每一种组蛋白各二个分子，形成一个组蛋白八聚体，约200 bp的DNA分子盘绕在组蛋白八聚体构成的核心结构外面，形成了一个核小体。这时染色质的压缩包装比(packing ratio)为6左右，即DNA 由伸展状态压缩了近6倍。200 bp DNA为平均长度；不同组织、不同类型的细胞，以及同一细胞里染色体的不同区段中，盘绕在组蛋白八聚体核心外面的DNA长度是不同的。如真菌的可以短到只有154 bp，而海胆精子的可以长达260bp，但一般的变动范围在180bp到200bp之间。在这200bp中，146 bp是直接盘绕在组蛋白八聚体核心外面，这些DNA不易被核酸酶消化，其余的DNA是用于连接下一个核小体。连接相邻2个核小体的DNA分子上结合了另一种组蛋白H1。组蛋白H1包含了一组密切相关的蛋白质，其数量相当于核心组蛋白的一半，所以很容易从染色质中抽提出来。所有的H1被除去后也不会影响到核小体的结构，这表明H1是位于蛋白质核心之外的。 3)染色体：在细胞的有丝分裂的分裂期由染色质经螺旋折叠形成，呈线状或棒状。 4) 有丝分裂：真核细胞的染色质凝集成染色体、复制的姐妹染色单体在纺锤丝的牵拉下分 向两极，从而产生两个染色体数和遗传性相同的子细胞核的一种细胞分裂类型。分裂具有周期性。即连续分裂的细胞，从一次分裂完成时开始，到下一次分裂完成时为止，为一个细胞周期。一个细胞周期包括两个阶段：分裂间期和分裂期，（这两个阶段所占的时间相差较大，一般分裂间期占细胞周期的90%-95%；分裂期大约占细胞周期的5%-10%。细胞种类不同，一个细胞周期的时间也不相同。）分裂期又分为分裂前期、分裂中期、分裂后期和分裂末期。细胞在分裂之前，必须进行一定的物质准备。细胞增殖包括物质准备和细胞分裂整个过程。有丝分裂是一个连续的过程按先后顺序划分为间期、前期、中期、后期和末期五个时期，在前期和中期之间有时还划分出一个前中期。 5) 分裂间期：主要完成DNA的复制和蛋白质的合成，DNA复制时边解旋编复制。 6) 姐妹染色单体：姐妹染色单体是指染色体在细胞有丝分裂（包括减数分裂）的间期进 行自我复制，形成由一个着丝点连接着的两条完全相同的染色单体。（若着丝点分裂，则就各自成为一条染色体了）。每条姐妹染色单体含1个DNA。 7) 同源染色体：二倍体细胞中染色体以成对的方式存在, 一条来自父本，一条来自母本， 且形态、大小相同，并在减数分裂前期相互配对的染色体。含相似的遗传信息。 8) 组蛋白：一组进化上非常保守的碱性蛋白质，其中碱性氨基酸(Arg，Lys)约占25%，存 在于真核生物染色质，分为5种类型(H1，H2A，H2B，H3，H4)，后4种各2个形成组蛋白八聚体，构成核小体的核心，占核小体质量的一半。组蛋白的基因非常保守。亲缘关系较远的种属中,四种组蛋白(H2A、H2B、H3、H4)氨基酸序列都非常相似。 9) 甲基化(methylation)：从活性甲基化合物(如S-腺苷基甲硫氨酸)上催化其甲基转移到其 他化合物的过程。可形成各种甲基化合物，或是对某些蛋白质或核酸等进行化学修饰形成甲基化产物。甲基化是蛋白质和核酸的一种重要的修饰，调节基因的表达和关闭，与癌症、衰老、老年痴呆等许多疾病密切相关，是表观遗传学的重要研究内容之一。最常见的甲基化修饰有DNA甲基化和组蛋白甲基化。DNA甲基化是指生物体在DNA甲基转移酶(DNA methyltransferase，DMT) 的催化下，以s-腺苷甲硫氨酸(SAM)为甲基
角朊细胞 角朊细胞的增殖和分化是一个受到精细调节的过程，并伴随着一系列形态学和生化改变，最终形成角质细胞，这就必然涉及到许多结构基因的同时活化与灭活，即基因表达的调控，而转录水平的调控尤为重要。现已发现许多转录因子如AP1、AP2、Sp1、POU结构域及C/EBP等可调节角朊细胞基因的表达。 目录
转录水平、翻译水平及翻译后水平，其中最常见的调控方式就是转录调控。现已发现AP1、AP2、NFκB、C/EBP、ets、Sp1及POU结构域等转录因子可作为表皮中的调控蛋白，从而调节编码套膜蛋白（involucrin, iNV）、转谷氨酰胺酶（transglutaminase，TG）、SPRR2A、兜甲蛋白（loricrin）、角蛋白及BPAG1等蛋白的基因的表达。本文就与角朊细胞基因表达有关的转录因子作一简要综述。 编辑本段转录因子的一般特征 转录因子（transcription factor）是能与位于转录起始位点上游50～5000bp的顺式作用元件（cis-acting elements）、沉默子（silencer）或增强子（enhancer）结合并参与调节靶基因转录效率的一组蛋白，并能将来自细胞表面的信息传递至核内基因。转录因子通常有几个功能域，可分为DNA结合域、转录调控域及自身活性调控域，DNA结合域可与特定的DNA序列（一般长8～20bp）相互作用，使转录因子与靶基因结合起来，随之转录调控域就可发挥其激活或抑制作用，通常这些结构域在结构与功能上是独立分开的。不同的转录因子还可结合于紧密相邻的DNA序列而形成一种多聚体结构来调节基因表达，这种组合调控（combinatorial regulation）不论转录因子是否激活及其含量多少均可激活基于靶基因中特定转录因子结合位点的转录。除启动基础转录活性外，转录因子还能整合从细胞表面经信号转导途径传递而来的信号[2]。 编辑本段激活角朊细胞基因表达的转录因子 （一）AP1 AP1转录因子通常以jun(c-jun、junB、junD)与Fos（Fra-1、Fra-2、c-fos、fosB）家族成员组成的同源或异源二聚体表达其活性，即结合于5’-GTGAGCTCAG-3’序列。目前已知AP1位点对于编码角蛋白（K1、K5、 K6及K19）、丝聚合蛋白原（profilaggrin）基因的最适转录活性十分重要[3,7]，编码角质化包膜（cornified envelope）相关蛋白-TG1、兜甲蛋白及INV的基因也含有功能性AP1 位点[8，9]，如hINV基因启动子在其转录起始位点上游2.5kb内有5个AP1共有结合位点（AP1-1～5），其中2个AP1位点AP1-1和AP1-5若同时发生突变时角朊细胞的转录水平就可下降80%；佛波酯（TPA）则可使AP1与hINV启动子处AP1-1及AP1-5位点的结合能力增强10～100倍，后经点突变实验证实AP1-1和AP1-5位点可部分介导佛波酯（TPA）诱导的效应[10]。丝聚合蛋白原、K1、兜甲蛋白及K19基因中的AP1位点可活化转录[3,6,7]，
转录因子Sp1对肿瘤转移的调控 闫隆鑫1，刘波1*，任海军2* 摘要转录因子SP1（transcription factor Sp1，SP1）在人体细胞中普遍表达并参与调控细胞增殖、凋亡及胚胎发育等生理活动。实验证实SP1在肿瘤细胞中存在异常表达，并积极调控肺癌、胃癌、乳腺癌等肿瘤的转移、恶变，但对其参与肿瘤细胞转移的机制，尤其在不同细胞系中，SP1的表达量及蛋白修饰，对肿瘤转移各阶段的影响，还不甚明确。本文整理了近期关于SP1参与调控肿瘤转移的研究及SP1的协同调控因子，藉此探究SP1在肿瘤监测及治疗中的发展方向。 关键词：转录因子SP1; 肿瘤; 转移 0 引言 肿瘤细胞的转移意味着肿瘤恶性程度增加，大部分肿瘤患者在临床确诊时已经存在肿瘤转移，常规治疗死亡率极高；而一旦诊断为肿瘤，由于不能确定其是否转移，往往会接受过度治疗，因此有关肿瘤细胞转移活性的检测是肿瘤治疗的关键。良性肿瘤发展为转移性肿瘤至少包括四个相互关联的过程：肿瘤细胞表面黏附分子减少，肿瘤间粘附能力下降；原细胞间基质分解，并在肿瘤细胞诱导下重建适宜肿瘤生长的基质；肿瘤细胞变形并生长出伪足，通过血管、淋巴管迁移到特定的侵入点；肿瘤细胞分裂增殖，在新区域生成血管，成为肿瘤组织[1]。转录调控因子参与调控相关蛋白的表达，往往存在异常表达的情况，在肿瘤转移过程中起到至关重要的调控作用。因此对肿瘤中转录调控因子的检测，能够在早期判断肿瘤转移的情况并对肿瘤转移的活性加以抑制。 SP1是人体细胞中广泛存在的转录调控因子，属于Sp/KLF锌指家族，通常作为主要的GC盒转录激活因子参与调控目标基因的表达。SP1的羧基端含有三个锌指结构，能够特异性结合DNA启动子的GC盒；SP1蛋白中段为其活化区，参与调控目的基因表达以及与其他转录调控因子的结合[2]；氨基端有一蛋白水解位点，能够引起泛素化诱导的SP1分解[3]。SP1在结合到目的基因启动子的同时，可以招募其他调控因子及SP1本身参与表达调控，因而与DNA的结合能力、转录调控区活性以及SP1在细胞内的含量对SP1参与的调控有重要的意义[4, 5]。SP1在肿瘤初期大量积累并积极调控肿瘤发展的各个阶段[6]，过表达的SP1参与诱导肿瘤增殖、凋亡，但对肿瘤转移的调控，不同肿瘤细胞系对SP1过表达有着不同 基金项目：大连市卫生局医学研究课题（WSJ/KJC-01-JL-01）；中央高校基本科研业务费（DUT15LK16）； 作者单位：1.大连理工大学生物医学工程系，辽宁省大连市，116024；2.大连市友谊医院普外科，辽宁省大连市，116100； 通信作者：任海军，E-mail:renhaijun369@https://www.doczj.com/doc/dc4607693.html,; 刘波，E-mail: lbo@https://www.doczj.com/doc/dc4607693.html,; 作者简介：（1996-），男，本科，在读本科生，主要研究方向生物医学工程
转录因子 摘要：随着众多生物基因组计划的完成及其蛋白质组学研究的不断深入，人类步入了系统生物学时代。基因组计划的完成提供了大量的DNA内在信息，解析出基因组中可能存在的全部基因的阅读框架，因此，接下来研究基因的表达调控特别是转录调控就显得非常迫切。另一方面，蛋白组学研究的突飞猛进给我们描绘出了细胞的蛋白质表达谱和网络谱，接下来研究蛋白质与蛋白质，蛋白质与DNA的相互作用将成为现在及以后相当长一段时间内的研究主题。有生物学家认为，21世纪对人类最具有挑战性的生物学主题就是“基因的全基因组调控”和”细胞的全蛋白质的生理功能”这两大难题。 然而，转录因子是可与基因调控序列结合并调控基因转录的一类核蛋白，研究转录因子就是研究转录调控的分子机制，研究一种或一类特定的蛋白质分子与DNA的结合特性，研究与DNA结合的蛋白质分子是怎样调控基因转录等问题。转录因子的研究实际上已构成上述两大生物学难题的一个交叉点，因此，对转录因子的深入研究已是一件极其迫切而且重要的课题。 DNA转录及转录因子 定义 转录：是指以DNA为模板，在RNA聚合酶的作用下合成mRNA，将遗传信息从DNA分子上转移到mRNA分子上，这一过程成为转录。真核生物DNA的转录在细胞核中进行，原核生物的转录在细胞质的核质区
内进行。 转录单元 转录单元是一段以启动子开始至终止子结束的DNA序列。 转录起始（transcription initiation）：转录因子通过识别基因启动子上的特异顺式元件并募集多种蛋白质因子，形成具有RNA聚合酶活性的转录起始复合体，从转录起始位点启动转录的过程。 转录终止子（transcription terminator）：基因编码区下游使RNA聚合酶终止mRNA合成的密码子，是一种位于poly(A)位点下游，长度在几百碱基以内的结构。 终止子可分为两类。一类不依赖于蛋白质辅因子就能实现终止作用。另一类则依赖蛋白辅因子才能实现终止作用。这种蛋白质辅因子称为释放因子，通常又称ρ因子 转录因子：能够结合在某基因上游特异核苷酸序列上的蛋白质，活化后从胞质转位至胞核，通过识别和结合基因启动子区的顺式作用元件,启动和调控基因表达。 转录因子是转录起始过程中RNA聚合酶所需的辅助因子。真核生物基因在无转录因子时处于不表达状态，RNA聚合酶自身无法启动基因转录，只有当转录因子(蛋白质)结合在其识别的DNA序列上后，基因才开始表达。转录因子是结合在某基因上游特异核苷酸序列上的蛋白质，这些蛋白质能调控该基因的转录。转录因子可以调控核糖核酸聚合酶（RNA聚合酶）与DNA模板的结合。转录因子不单与DNA序列上的启动子结合，也可以和其它转录因子形成-转录因子聚合体，来影
植物转录因子蛋白质结构 转录因子是生物体内直接结合或间接作用于基因启动子区域、形成具有RNA聚合酶活性的转录复合体的蛋白质因子，通过其调控基因的表达来影响生物的表型及对外界刺激的保护，从而完成了生物在转录水平的调控。按功能可分为通用转录因子、序列特异性转录因子、辅助转录因子等。而与RNA聚合酶I、Ⅱ、Ⅲ相对应的有3类转录因子，分别是TFI、TFⅡ、TFⅢ。锌指蛋白就是属于其中的TFⅢ型转录因子，它是生物中发现种类最多、研究较为广泛、在真核生物中具有重要调控作用的一类转录因子。 通过对蛋白质的结构进行分析表明，典型的植物转录因子一般由DNA结合区(DNA—binding domain)、寡聚化位点(oligomerization site)、转录的调控区(transcription regulation domain)、细胞核定位信号区(nuclear localization signal，NLS)组成，这些功能区域决定了各个转录因子的具体功能。 DNA结合区(DNA—binding domain)DNA序列中有许多具有重要作用的顺式作用元件，能够识别并与之结合的氨基酸序列就是转录因子的DNA结合区。相同类型的转录因子都能够识别比较保守的氨基酸序列(DNA结合区)。而且植物转录因子的分类依据就是DNA结合区和寡聚化位点的保守区的差异。其中bHLH结构域、bZIP结构域、锌指结构域、MADS结构域、MYC 结构域、MYB结构域和类Myc蛋白等都是典型的植物转录因子的DNA结合区。这些典型的结合区与顺式作用元件识别及结合的特异性由DNA结合区中特定的氨基酸序列来决定。它们与顺式作用元件的亲和性和特异性由DNA结合区的二级结构来决定。 bHLH(basichelix-loop-helix)家族转录因子普遍存在于真核生物中。目前,已在拟南芥中发现了147个bHLH家族转录因子基因。bHLH转录因子约由60个氨基酸残基组成,因HLH结构上游富含碱性氨基酸而得名,含有两个相连的基本亚区,即HLH Motif及其上游富含碱性氨基酸基序,其中碱性氨基酸基序与DNA结合有关,对基因的转录发挥调控作用。bHLH转录因子的HLH 区长为40-50个氨基酸残基,参与二聚体形成,有HLH蛋白的共同模体,即具有两条短小的既亲水又亲脂的两性α-螺旋,螺旋区的长度为15-16个氨基酸,含有各种保守的氨基酸残基,两个α-螺旋由连接区(环)相连,连接环的长度不等,由12-28个氨基酸组成,螺旋的一侧有疏水氨基酸。bHLH转录因子两条α-链依赖疏水氨基酸的相互作用形成同型或异型二聚体,从而与启动子的不同部位相结合。缺少碱性区的HLH蛋白可以与bHLH蛋白形成二聚体,但无结合DNA 的能力。 bZIP转录因子是真核生物转录因子中分布最广泛、最保守的一类转录因子。几乎所有真核细胞中都发现了bZIP结构域的转录冈子。根据植物bZlP转录因子结构特点和功能可以将bZIP 家族划分为10个亚族。所有的bZIP转录因子除了都具有两种保守的结构域外，同一个亚族内的bZIP转录因子还有额外的共有特征，如亮氨酸拉链的大小、类似的DNA结 合碱性结构域和类似的cis元件等。植物bZIP类转录因子的共同结构特点是：(1)含有与特异DNA序列相结合的碱性结构域，大约由20个氨基酸组成，紧靠亮氨酸拉链结构域的N末端，能与专一的DNA序列进行相互作用；(2)参与寡聚化作用的亮氨酸拉链区与碱性区紧密相连，每7个氨基酸的第7位含有一个亮氨酸。亮氨酸拉链形成一个两亲的螺旋结构，该结构参与bZIP蛋白与DNA结合之前的二聚体化；(3)转录因子的N末端含有酸性激活区；(4)以二聚体形式结合DNA，肽链N末端的碱性区与DNA直接结合。 至今，发现了三类锌指结构。一类是类似TFIIIA，如哺乳动物细胞的SP1。第二类锌指结构是通过NMR（核磁共振）检测到的，这类结构有点类似于HTH结构。它是由两个环-螺旋结构组成，命名为“双环-锌-螺旋”（double loop-Zn-helix），锌离子与在环开始部分中的两个半胱氨酸和两个а-螺旋的N端的两个氨基酸残基作用，靠近第一个а-螺旋N端的残基决定了
转录因子 转录因子是细胞的蛋白质哨兵，它决定DNA 中众多基因中的某些特定基因在给定的时间内转录为mRNA 。细菌里面含有200~300种转录因子，而动物细胞包约含1000种。通过使DNA 和基本转录装置联系起来，转录因子决定了细胞的蛋白质结构。作为初级控制者，它们在细胞内浓度很低。其浓度很大程度上取决于具体的蛋白质、细胞类型和环境因素，根据经验法则，它的在浓度在n 摩尔(nM)浓度范围，细菌的每个细胞有1~1000个转录因子，在哺乳动物细胞中约有36 10~10个。通常，低浓度转录因子只控制少数基因，高浓度的则相反。 转录因子激活DNA 转录 拓展：在分子生物学中，转录因子（Transcription factor ）是指能够结合在某基因上游特异核苷酸序列上的蛋白质，这些蛋白质能调控其基因的转录。转录因子可以调控核糖核酸聚合酶（RNA 聚合酶，或叫RNA 合成酶）与DNA 模板的结合。转录因子一般有不同的功能区域，如DNA 结合结构域与效应结构域。转录因子不单与基因上游的启动子区域结合，也可以和其它转录因子形成转录因子复合体来影响基因的转录。 转录因子的调节是一个十分复杂的过程, 因为它取决于很多因素，其中最明显的是其他的DNA 结合蛋白(包括转录因RNA 聚合酶 转因录 子
子等)以及局部的染色体结构. 早期的体外实验认为DNA序列决定转录因子的装配顺序，但愈来愈多的证据显示转录的激活取决于大量的转录因子的相互作用。目前表观遗传学似乎对转录激活也扮演重要角色。 通常每个细胞只含大约10个四聚体，大多数转录因子有相似或者更高的浓度为每nM几十个或上百个。有趣的是，DNA非特定的吸引力使90%的乳糖抑制体被吸附在DNA周围，只有少数的溶解在细胞质内。这引发了一个重要的问题：与如此少量的随机波动是如何被生物细胞控制的？例如，如果这些转录因子是完全随机的，在细胞分裂时，如此少量的的转录因子可能使某些子细胞完全不含转录因子。 更多的的努力被投向了一直以来研究最多的蛋白质，如p53 ——一种出现在近50%的癌症中的转录因子。正如其他许多蛋白质一样，它的名字起源于它的最初表征凝胶，p53蛋白的分子量为53kDa。现在我们已经知道它的质量为43.7kDa，它缓慢的移动速度是笨重的脯氨酸残基造成的，但它的名字p53还是保留了下来。这些转录因子促使细胞程序性死亡来防止其继续增殖，抑制肿瘤的生长。它有自己的特征浓度约100 nM。转录因子通过与来自受体信号相互作用，来改变它们与DNA的结合属性，从而调整转录信息。癌细胞中DNA的变异改变p53与它控制的下游基因的结合属性，从而阻止细胞死亡，导致细胞不可控增殖。 肿瘤蛋白p53——P53与DNA结合