The polymorpholism of DOF genes expressed in these plants and their possible roles remain to be investigated.
In a cDNA library of developing seeds from peanut variety 063103, eight clones of DOF cDNAs from over 20,000 clones have been found. These clones were sequenced, translated and compared with AtDofs, GmDofs and ZmPBF. The AhDof proteins are structurally classified and their functions are suggested via bioinformatic and theoretic analysis.
2. MATERIAL AND METHODS
Eight DOF cDNA clones are from a cDNA library of developing seeds of peanut line 06-3103, which is leaf spot disease resistance. Additional DOF cDNA sequences similar to DF cDNAs from peanut seed are screened from cDNA libraries of leave and root of control and innoculated with Ralstonia solanacearum. The vector is pDNR-LIB (CLONTECH). The plasmids are purified and the inserts are examined by PCR with M13F and M13R primers. The DOF clones are sequenced with M13R and T7 primers by BGI (Huada Gene) Company in Wuhan. The DOF ESTs from peanut cultivars VBL6, GT-C20 of developing seeds are obtained with blastn-est program from GeneBank . The cDNA sequences are translated into protein sequence with AUGUSTUS (version 2.1) . The AhDof ests from 06-3103 are deposited in GeneBank as: GW391722, GW391723, GW391- 724, GW391725, GW391726, GW391727, GW391728, GW391729.
Other proteins and Ests from peanut listed in text are from GeneBank and Swiss-Prot. DNA extracted from Material listed in Table 3.
2.2. Cloning of DOF Genes from Genomic DNA of Nine Peanut Cultivars
DNA was extracted with CTAB method  and used as templete in PCR cloning of DOF genes.
PCR primers for GW391729 are: S37: 5’GAAGCAGAAGGAGGAAGG 3’, A1133: 5’ACAACAAAGCAAAGTAATAAA 3’.
PCR reaction mix was 25 μL in total, contained ddH2O 18.3 μL 10x PCR buffer 2.5 μL, dNTP (each 10 μM) 1 μL, forward and reverse primers (10 μM) each 1 μL, DNA templete 1 μL, Taq polymerase (Takara) 0.2 μL. The PCR reaction for GW391729 is: 95˚C 2 min, then the cycle 95˚C 40 s, 49.5 (each cycle down 0.4)˚C 30 s, 72˚C 40 s, for 20 cycles, next cycle, 95˚C 40 s, 41.5˚C 30 s, 72˚C 40 s for 18 cycles, then 72˚C for 7 mins. The specific products are purified and constructed into pMD18T vectors, the positive clones are sequenced by BGI (Huada) genomic company and Ebioe (Shenggong) company.
2.3. Sequence Comparison and Analysis
DOF cDNAs from peanut line 06-3103 were batch blasted with blasx-nr and blast-swiss to get mostly similar DOF proteins from arabidopsis and soybean, maize PBF was used as a reference . Then the selected seed DOF proteins were pairewise compared with GeneDoc software. The conserved domains are searched with conserved motifs listed by Lijavetzky et al. . The DOF proteins are classified by the similarity comparison with that of arabdopsis according to Yanagisawa .
2.4. Bioinformatic Analysis on Function of DOF Proteins
The information of expression patterns of AtDofs similar to Ahdofs from developing seeds of 06-3101 were collected from iHOP (Information Hyperlinked over Proteins), http://www.ihop-net.org/ [40,41]. The information about functions of related DOF proteins are also picked from literature by searching key word “DOF” and genebank association numbers in NCBI Pubmed, and by searching TAIR and linked information.
3.1. Eight Different DOF Genes Expressed in Developing Peanut Seed
Eight different DOF genes were found from a cDNA library from developing seed with over 20,000 clones of cultivar 063103. The resulted DOF proteins belong to DOF type II, III, IV, V, and VII respectively according to Lijavetzky et al.  (Table 1). GW391725, GW391726 and GW391727 are similar to three CYCLING DOF FACTORs (CDF) genes (type II), AtCDF3, AtCDF2 and AtCDF1 in arabidopsis respectively (Figure 1(a)). GW- 391722 belong to type III, similar to GmDof 11 and AtDof2.1 (Figure 1(b)). GW391723 is similar to AtDof2.2
Table 1. Comparison of the conserved domains between AhDofs, GmDof and AtDofs besides DOF domains.
(Figure 1(c)), belong to type V. GW391724 is similar to AtDof3.4 and GmDof2 (Figure 1(d)), belong to type IV. GW391728 is similar to AhDof3 (ACF74278.1) and GmDof5 (Figure 1(e)), belong to type VII; GW391729 is similar to but at less similar level to AtDof2.2 and GmDof12 (Figure 1(f)). Among these similar AtDof and GmDof proteins, All DOFs are expressed in developing seed [19,40].
Different types of DOF proteins not only have their distinct sequences in N and C terminal regions (Table 1), but also have their specific characteristics in conserved DOF finger regions (Figure 2). The key structure in zinc finger domain is two CXXC bridges and a C residue surrounded by some conserved amino acids between two CXXCs. Four C residues in two CXXCs binding with one zinc ion cause the formation of a finger like conformation in this region. There are four variable motifs in zinc finger domain among AhDofs, GmDof and AtDofs. The first motifs is N (or K/D/E) SM (orT/P/S) D (E/N) which locates right after CPRC (Figure 2). The second is NVN (NIN/NFS/NYS/SLS/SLT), which is after KFCYYNNY, the middle conserved domain containing middle C residue (Figure 2). The third variable region cover the second CXXC, H (or/Y) FACKN (or/A/S/T/K) CQ (or/R/K) RYWTS (or/A/H/K/R) (Figure 2). The forth variable region is AGR (/GGS/GVS/GGC/GSY) (Figure 2), right before the possible NLS sequence RKN—KR [42,43]. The N terminal CXXC is conserved as CPRC in AhDofs listed, the second CXXC in AhDofs listed have variation as CKNC (CDFs), CKSC (GW391724, GW- 391722), CKAC (GW391729, GW391728, and PBF of Maize) and CKTC (GW391723) (Figure 2).
It is obvious that in all CDFs there is coherence among four variable regions and consistency among different CDFs in the same region (Figure 2). It is coherent that the four variable regions are all relatively conserved in CDFs. They are N (or K) SMD (for peanut) (E for arabidopsis), NV (or I) N, HFCKN (for peanut and soybean) (A or K for arabidopsis) Q—A(or S), AGR. The CDFs are consistent in that the conserved combination of amino acids are rich of N or Q (only one) which are polar amino acid residues without charge. In the key variable region, the variable amino acids have the same physiochemical property, such as D and E in the first region, V and I in the second region (Figure 2). Some are restrictly the same, such as M before D or E, N-N, Q after the second CXXC, AGR before RKN. The common property in CDF zinc DOF domain right before nuclear
Figure 1. Phylogenetic trees of AhDofs with their similar DOF proteins.
Figure 2. Comparison of DOF domain sequences between similar AhDofs, AtDofs and GmDofs.
localization sequences possibly provided the structure basis for their common specific DNA binding property related to the function of CDF types of DOF proteins.
The second similarity group of DOFs contains GW- 391724, At5g66940 and GmDof2. They have variable I region as D(E)ST, variable II region as NFS, the third variable region as SCR—H, the forth variable region as G(V)SR (Figure 2).
The specific conserved domain in GW391722 similarity group is in region III, YFCKSCRRYWTK (Figure 2).
GW391722,23,28,29 all contained a same N-glycosylation site overlay the DOF middle conserved domain and variable II region (NYSL), All three CDF and GW- 391724 do not have N-glycosylation site in conserved DOF domain, but three overlayed N-glycosylation site on the N-terminal outside of conserved DOF domain.
From ESTs deposited in GeneBank database derived from various cDNA libraries of developing seed of peanut, partial sequences of four DOF cDNAs (GO332017, GO339137, GO324523, GO332293) from VBL6 cDNA library, two DOF cDNAs (EE124644, EE125478) from Luhua4 cDNA library, and one (ES703897) from Tifton C20R5 cDNA library were obtained, translated and compared with DOF proteins from 06-3103. GO332017 is a partial sequence with 215 amino acids, highly similar to C terminal domain of AhCDF3s (GW391726, GW- 391725) (Figure 3(c)). Within its N terminal 205 amino acids residues, only 13 amino acids are different from that of GW391726, within its N terminal 86 amino acids residues, only 9 amino acids are different from that of GW391725 (Figure 3(c)), which is different between species. GO339137 is highly similar to a 67 aa at C terminal of GW391722, with only 6 aa residues difference between them (Figure 3(a)). These differences are possibly the result from differences between species. GO- 324523, GO332293 have little similarities to other domains outside of DOF zinc finger domain with known AhDofs.
3.2. GW391729 Was Expressed in Peanut Leave Inoculated with Ralstonia solanacearum
DOF genes expressed from cDNA libraries of roots and leave with control and inoculated with Ralstonia solanacearum from same cultivar 063103 were examined. GW- 391729 was found expressed only in leave inoculated with Ralstonia solanacearum, not in controled leave (Figure 4), this indicates that GW391729 is possibly related to the leaf resistant to Ralstonia solanacearum. In leave of both controled and inoculated with Ralstonia solanacearum, similar but different ests to GW391724
Figure 3. Alignment of AhDOFs and AtDOFs.
Figure 4. Comparison of GW391729 and cDNA F09 from leaf inoculated with Ralstonia solanacearum.
were expressed (Figure 5), these ests are not the same DOF proteins as GW391724, possibly related to the leaf development. In root inoculated with Ralstonia solanacearum, an est similar to but different from GW391722 was expressed (Figure 6), its function is possibly related to root response to Ralstonia solanacearum. As for function of GW391724 and GW391722 expressed in seed and different from those in leaf and root respectively, need further investigation.
3.3. Evolution Origion of AhDofs from Developing Seeds
The blastn results using the est sequences of AhDofs from developing seeds of 063103 with Genebank tools show that the most similar AhDofs are from wild peanut species A. ipaensis (Table 2), which is the source of BB genome of cultured peanut specie . This indicates that all the eight AhDofs are possible from peanut BB genome.
Among AhDofs, ESTs from wild Arachis species similar to GW391722, GW391725, GW391726, GW- 391727 are only from A. ipaensis (Table 2), indicates these four genes are possible locate in only BB genome. For GW391723, GW391728, GW391729, beside that the most similar ests are from A. ipaensis, there are two similar ests to each from seed of A. duranensis (Table 2), which is the origin of AA genome of cultured peanut
Figure 5. Comparison of GW391724 and similar cDNAs from leaf inoculated with Ralstonia solanacearum.
Figure 6. Comparison of GW391722 and similar cDNAs from root inoculated with Ralstonia solanacearum.
specie . This reveals that in AA genome there are possible similar alle genes.
3.4. GW391729 Are Different between Strains with Different Seed Numbers in a Fruit
Sequences of GW391729 from 8 cultivars were compared and analysied. Most of cultivars with 3 seeds per fruits have distinctive sequences, which is different from those with 2 seeds per seeds (Table 3). But it seems that size of seeds and resistance to intrusion of seed do not related to GW- 391729.
The integrated function of a protein is determined by its integration of structure and sequence. The different domain of a protein may have different activities. DOF proteins have relatively conserved zinc finger domain to bind to DNA promoter sequence, and variable C terminals differring at amino acid composition and lengths. The different structure in zinc finger domain determined their specific binding targets . Specific C terminal sequences and structure determined their specific reaction other than binding to DNA. Thus the specific function of
Table 2. Similarity analysis of DOF est to those from original species in Arachis genus.
Table 3. Differential sites of GW391729 among from 8 cultivars.
DOF proteins can be deduced by their sequence similarities to each structure domain with known function.
GW391725, GW391726 and GW391727 are three highly similar DOFs within AhDofs. They are mostly similar to AtCDF1 (At5g62430), AtCDF2 (At5g39660) and AtCDF3 (At3g47500). They contain all the conserved motifs of group II DOF proteins  (Table 1). CDF genes were first identified in Arabidopsis with yeast two-hybrid screen for proteins interacting with C terminal kelch repeats of FKF1, as well as that of LKP2, which can degrade CDFs, but not that of ZEITLUPE (ZTL) which functions in different signal transduction pathways [45,46]. Three AtCDFs were isolated as AtCDF1 (At5g62430), AtCDF2 (At5g39660), AtCDF3 (At- 3g47500) [45,46]. AtCDF1 was further found to bind to the promoter of CO gene as a key negative factor in flowering pathway. By degrading the CDF1, FKF1 can release the inhibition of CDF1 on CO expression and thus release the inhibition of flowering [45,46]. Thus AtCDF1 is an inhibitor for flowering. The highest expression of AtCDF1 is in cauline senescent leave, stem between 1st and 2nd intenode, the intermediate expression level is detected in vegetative rosette, early embryo stages 3 to 5, and the pods in the same stages, each flower organs of flower stages 12 to 15, as well in mature pollen. AtCDF1 was also reported restricted in vasculature . AtCDF2 and AtCDF3 do not have effect on flowering , while another study indicates AtCDF2 also function in repression of photoperiod induced flowering . AtCDF2 and AtCDF3 expressed more specifically in developing seeds, the expression level increased from torpedo stage till the highest level at curling cotyledon stage 9 [40,41]. The expression of AtCDF2 is more specific in developing seed and dry seeds than AtCDF3. Besides in developing seeds, AtCDF2 is also expressed in mature pollen and cauline senescent leave. AtCDF3 expressed at higher level in cauline senescent leave, stem between 1st and 2nd intenode than AtCDF2, it also expressed in vegetative leave, stage 15 flower/ pedicel, stage 15 petal and stamen, lower expression lever in sepal, early stage of surface of pods [40,41]. All the three AtCDFs (At3g47500, At5g39660 and At5g62430) are expressed in sperm cells and they are the only DOF genes expressed there of the 32 DOF transcription factors . In cold acclimations, AtCDF2 and AtCDF3 are two of five DOF genes upregulated [29,30]. AtCDF3 has been known as H-protein promoter binding factor-2a. Since three AtCDFs all interact with FKF1 and LPK, and they all have long N terminal domains before DOF zinc domains which located just before NLS and binding to DNA sequences on promoters of genes, thus may cause large bulk at binding sites and possibly hinder the interaction of other transcription factors with the promoters, so that the function of CDFs may be conserved in higher plants, and they act as inhibitors for expression of certain genes.
GW391722 has a similarity of 94% and 91% to At- 5g60200 and At2g28510 within 56 aa residues in DOF conserved domain respectively, 85% to GmDof13 in DOF domain with 97 aa residues, 54% to GmDof11 in DOF zinc finger containing domain with145 aa residues, it also have DOF group III conserved motif 13, 11, 20 , belonging to group III. Both group III DOF At5g60200 and At2g28510 were reported to regulate vascular formation process . At2g28510 was upregulated by cytokinin  and GA4  stimulation. Compared to At2g28510, more expression of At5g60200 was detected in shoot meristem [39,40]. The expression of At2g28510 enriched in 24 h imbibed seeds. In G. max, under control of 35S CaMV promoter, GmDof11 increase the lipid content in seeds of trangenic Arabidopsis plants by binding to a P-box like cis DNA element and also regulate the expression of a set of genes . This indicates that GW- 391722 may have possible function in both vascular differentiation and metabolism by binding to promoters of a set of genes.
GW391724 shared sequence similarity of 81% to GmDof2 zinc finger domain with 64 aa residues, 73% to AtDof3.4 (At3g50410, OBP1) zinc finger domain with 63 aa residues, 72% to AtDof5.8 (At5g66940) zinc finger domain with 66 aa residues. It has a DOF group IV conserved motif 33 and a undetermined motif among GW- 391724, AtDof5.8, and GmDof2 (Table 1), so possibly belong to group IV . Group IV DOF At5g66940 is highly expressed in shoot apex inflorescence, vegetative, and transition stage, and in a patchy pattern in young floral organ primordia, latter stages limited to stamens and carpels, and early stages of developing seeds [39,40]. At5g66940 was down regulated in the strubbelig-like mutant (slm) in which plant organogenesis controlled by SLM genes, was one of genes responsive to SLM genes . AtOBP1 was recently reported upregulating cell cycle specific genes, shortening G1 phase and overall length of the cell cycle, function as specific cell division regulator .
Among AhDofs, GW391728 shared the highest similarity to PBF at N terminal sequences including zinc finger domain (65%, 55 out of 84 aa), which belong to group VII , thus GW391728 is also possibly a cis DNA element P box-like binding factor. Besides, GW- 391728 has the highest similarity to ACF74278/ DQ- 889513 from peanut line Luhua14 with sequence identity of 169/181 aa (93%), but is much less similar to protein sequence of GO324523 from peanut line Tifrunner (Figure 2(b)), and did not found similar ests in peanut GTC20R or other cultivars in genebank database. Comparison of protein sequences from eight cultivars reveals that GW391729 is possible related to the seed number in fruits. But it is only display such phenomenon among 8 evolutionally closed cultivars. GW391729 is also has possible function on leaf disease resistance, thus its function may related to development and possible cooperate with other DOF proteins.
The protein sequences of GW391723 and GW391729 shared similarities to that of At2G28810 with 48% at 76 aa/157aa and 59% at 61aa/103 aa respectively. Amino sequence of GW391723 has a conserved group V motif 17 at N-termial domain (Table 1) and possibly belong to group V , but which of GW391729 does not have similar sequence in other domains besides DOF zinc finger domain with known DOF proteins. Group V At2G28810 expressed in all stages but not in late pod stages [39,40]. It is preferred expressed in phloem companion cells. Thus, it has a possible role in differentiation of vascular tissue .
This paper was supported by Natural Science Foundation of Hubei province (Bzy05011) and 863 project (2006AA10A114).