IPA1

IDEAL PLANT ARCHITECTURE1 (IPA1), a pleiotropic gene isolated through a map-based cloning approach, has been shown to be one of the key regulators that determine plant architecture.

Annotated Information

Figure 1 Figure 1. IPA1 Directly and Positively Regulates the Expression of Os TB1. (A) IPA1 binding profile in the promoter of Os TB1. The open arrowhead refers to the GTAC around the peak summit and the solid arrowhead to the GTAC around the subsummit. The red vertical line denotes the peak summit. The primer pair Os TB1 Pro (see Supplemental Table 1 online) was used for amplifying the Os TB1 promoter. (B) Validation of IPA1 direct binding sites in the Os TB1 promoter by ChIP-qPCR analysis. The fold enrichment was normalized against the promoter of Ubiquitin. aGFP, antibodies against GFP. Values are means 6 SE (n = 3). The double asterisks represent significant difference determined by the Student’s t test at P < 0.01. (C) EMSA showing that GST-IPA1 could directly bind to the promoter of Os TB1. The 10- and 40-fold excess nonlabeled or mutated probes were used for competition. (D) Comparison of Os TB1 expression levels between NIL-IPA1 and NIL-ipa1 in SAs and YPs, respectively. Rice Actin was used as reference. Values are means 6 SE (n = 3). The double asterisks represent significant difference determined by the Student’s t test at P < 0.01. (E) Suppression of tiller number by tb1/fc1 in RIL-ipa1/tb1 lines. Bar = 20 cm. (from reference)

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Figure 2Figure 2. IPA1 Directly Binds to the DEP1 Promoter. (A) IPA1 binding profile in the promoter of DEP1. The open arrowhead refers to the GTAC around the peak summit, solid arrowheads to other GTAC sequences around subsummits, and red vertical lines to peak summits. Primer pairs DEP1-Pro1 and DEP1-Pro2 were used for amplifying DEP1 Pro-1 and DEP1 pro-2,respectively. (B) Validation of IPA1 direct binding sites in the DEP1 promoter by ChIPqPCR analysis. The fold enrichment was normalized against the promoter of Ubiquitin. aGFP, antibodies against GFP. Values are means 6 SE (n = 3). The double asterisks represent significance difference determined by the Student’s t test at P < 0.01. (C) Direct binding of IPA1 to the DEP1 promoter in EMSA. The 10- and 40-fold excess nonlabeled or mutated probes were used for competition. (from reference)

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Figure 3 Figure 3. DEP1 Is Positively Regulated by IPA1. (A) Suppression of plant height by dep1 in RIL-ipa1/dep1 lines. Bar = 20 cm. (B) Statistical analysis of (A). Values are means 6 SE (n = 15). Different lowercase letters indicate significant differences between different genotypes (P < 0.01, one-way analysis of variance). (C) Suppression of panicle length by dep1 in RIL-ipa1/dep1 lines. Bar = 5 cm. (D) Statistical analysis of (C). Values are means 6 SE (n = 15). Different lowercase letters indicate significant differences among different genotypes (P < 0.01, one-way analysis of variance). (E) Expression patterns of IPA1 and DEP1 in different tissues revealed by qPCR analyses. R, roots; C, culms; B, leaf blades; S, leaf sheathes; A, SAs; Y, YPs; M, mature panicles. Rice Ubiquitin was used as reference. Values are means 6 SE (n = 3). (F) Comparison of DEP1 expression levels between NIL-IPA1 and NIL-ipa1 in SAs and YPs, respectively. Rice Actin was used as reference. Values are means 6 SE (n = 3). The double asterisks represent significant difference determined by the Student’s t test at P < 0.01. (from reference)

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Function

IPA1 Suppresses Rice Tillering Mainly through Positive Regulation of Os TB1

Os TB1 acts as a negative regulator of rice tillering (Takeda et al., 2003; Minakuchi et al., 2010) and that Os TB1 is a potential direct target of IPA1. The peak summits for IPA1 binding sites were located 96 and 187 bp upstream of the Os TB1 TSS in the two YP replicates, respectively.However, the Os TB1 promoter was significantly enriched in both SAs and YPs by the ChIP-qPCR analysis, indicating that Os TB1 is probably under the control of IPA1 in both tissues (Figure 1B). Further analysis of binding peaks revealed that the GTAC motif, rather than the TGGGCC/T motif, existed in the Os TB1 promoter. To test whether IPA1 can directly bind to the GTAC motif in the Os TB1 promoter, we performed an EMSA assay and found that GST-IPA1 could bind to a 59-bp probe from the Os TB1 promoter (Figure 1C). The mutation of GTAC to ATAC abolished its binding affinity, demonstrating that the binding was dependent on the GTAC motif. As a negative regulator in rice tiller development, Os TB1 is expressed in axillary buds, and its deficiency results in a semidwarf phenotype with a significant increase in tiller number (Takeda et al., 2003; Minakuchi et al., 2010). This suggests that the reduced tiller phenotype of ipa1 plants may result from the direct activation of Os TB1 by IPA1. The RNA level of Os TB1 was significantly upregulated in SAs in NIL-ipa1, but much less significantly in YPs (Figure 1 D). Furthermore, the double mutant analysis showed that the mutation of Os TB1 could suppress the tillering phenotype of ipa1 (Figure 1E). Since Os TB1 is homologous to PCF1 and PCF2, we tested whether Os TB1 could also interact with IPA1. A weak interaction was detected in the yeast two-hybrid assay , and EMSA further showed that Os TB1 could directly bind the TGGGCC/T motif, indicating that Os TB1 also functions as an adaptor for the interaction between IPA1 and the TGGGCC/T motif. Considering that Os TB1 is not only a target of IPA1, but also can interact with IPA1, we propose that Os TB1 is probably involved in the feedback regulation of IPA1 transcriptional activity.

Increased Plant Height and Panicle Branches

DEP1 is another important regulatory gene that affects rice architecture, especially panicle morphology. A truncated mutation of DEP1 enhances panicle meristematic activity, resulting in reduced length of inflorescence internodes, increased number of grains per panicle, and a consequent increase in grain yield (Huang et al., 2009). IPA1 ChIP-seq analysis revealed that DEP1 was also a direct target of IPA1 . The peak summits of IPA1 binding were located 566, 514, 440, and 500 bp upstream of the DEP1 TSS in SA rep 1, SA rep 2, YP rep 1, and YP rep 2, respectively (Figure 2A). Within the peaks in the DEP1 promoter, besides the highest peak summit, there are other two subsummits showing local max sequencing reads (Figure 2A). Through analyzing the DEP1 promoter sequence, several GTAC motifs were found, and three sites around the peak summits and subsummits were considered to be responsible for the binding of IPA1. To confirm this, ChIP-qPCR was performed. As shown in Figure 2B, the two specific regions were significantly enriched. We then tested whether IPA1 could directly bind to the promoter of DEP1 by EMSA with three 59-bp sequences around the peak summit and subsummits as probes and found that IPA1 could bind to all the three probes but could not bind to a probe containing the mutated binding core motif (Figure 2C). These results demonstrate that IPA1 can directly bind to the promoter of DEP1 at different sites and the GTAC core motifs are responsible for IPA1 binding, suggesting that DEP1 is a direct target of IPA1. To further understand the biological functions of DEP1 as a direct target of IPA1, we generated recombinant inbred lines by crossing Ri22, an ipa1-carrying cultivar (Jiao et al., 2010), with LJ5, a rice variety carrying the dep1 mutation (Huang et al.2009). As shown in Figures 3A and3B, the plant height of the RIL-ipa1/dep1 lines was reduced to a similar level to the RIL-IPA1/dep1 lines. Consistent with the previous result that dep1 does not affect rice tillering, the tiller number of RIL-ipa1/dep1 lines exhibited no significant difference from RIL-ipa1/DEP1 lines. We also noticed that the panicle morphology of the RIL-ipa1/dep1 lines was dense and erect (Figures 3C and 3D). Compared with the RIL-ipa1/DEP1 lines, the panicle length of RIL-ipa1/dep1 lines was significantly reduced, but no obvious change in panicle branches was found. Like IPA1, DEP1 was also strongly expressed in culms, SAs, and YPs, but weakly in roots (Figure 3E), suggesting that IPA1 may regulate the expression of DEP1. We further examined the expression of DEP1 in NIL-ipa1 lines and found that DEP1 transcripts were significantly increased in the SA of NIL-ipa1 lines, but only slightly in YP (Figure 3F). Therefore, it is likely that IPA1 functions as a positive regulator of DEP1 in regulating plant height and panicle length in rice.

Protein

IPA1 encodes the protein Os SPL14, and in the ipa1 mutant, one nucleotide substitution located in the recognition site for microRNA156 (miRNA156) perturbs IPA1 mRNA degradation, which results in accumulation of IPA1 and leads to the formation of ideal plant architecture with decreased tiller number and increased plant height and panicle branches (from reference).

WEALTHY FARMER’S PANICLE, another overexpression allele of Os SPL14, resulted from an epigenetic change in the Os SPL14 promoter and shows a similar phenotype (Miura et al., 2010). Therefore, it has been suggested that Os SPL14 alleles have great potential for breeding (Jiao et al., 2010; Miura et al., 2010).

Labs working on this gene

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China(Zefu Lu, Hong Yu, Yonghong Wang, Jiayang Li) State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China(Xingming Hu, Qian Qian) State Key Laboratory of Plant Cell and Chromosome Engineering and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China(Xiangdong Fu)

References

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Structured Information

LOCUS GU136674 7229 bp DNA linear PLN 29-JUN-2010 DEFINITION Oryza sativa Japonica Group IPA1 (IPA1) gene, complete cds. ACCESSION GU136674 VERSION GU136674.1 GI:299482811 KEYWORDS . SOURCE Oryza sativa (rice)

 ORGANISM  Oryza sativa
           Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
           Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
           clade; Ehrhartoideae; Oryzeae; Oryza.

REFERENCE 1 (bases 1 to 7229)

 AUTHORS   Jiao,Y., Wang,Y., Xue,D., Wang,J., Yan,M., Liu,G., Dong,G.,
           Zeng,D., Lu,Z., Zhu,X., Qian,Q. and Li,J.
 TITLE     Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture
           in rice
 JOURNAL   Nat. Genet. 42 (6), 541-544 (2010)
  PUBMED   20495565

REFERENCE 2 (bases 1 to 7229)

 AUTHORS   Jiao,Y., Wang,Y., Qian,Q. and Li,J.
 TITLE     Direct Submission
 JOURNAL   Submitted (22-OCT-2009) State Key Laboratory of Plant Genomics and
           National Center for Plant Gene Research, Institute of Genetics and
           Developmental Biology, Chinese Academy of Sciences, No.1 West
           Beichen Road, Chaoyang District, Beijing 100101, China

FEATURES Location/Qualifiers

    source          1..7229
                    /organism="Oryza sativa"
                    /mol_type="genomic DNA"
                    /db_xref="taxon:4530"
                    /chromosome="8"
                    /map="RM149-RM1345"
    gene            995..5150
                    /gene="IPA1"
    mRNA            join(995..1566,3997..4130,4233..5150)
                    /gene="IPA1"
                    /product="IPA1"
    CDS             join(1118..1566,3997..4130,4233..4903)
                    /gene="IPA1"
                    /note="transcription factor"
                    /codon_start=1
                    /product="IPA1"
                    /protein_id="ADJ19220.1"
                    /db_xref="GI:299482812"
                    /translation="MEMASGGGAAAAAGGGVGGSGGGGGGGDEHRQLHGLKFGKKIYF
                    EDAAAAAGGGGTGSGSGSASAAPPSSSSKAAGGGRGGGGKNKGKGVAAAAPPPPPPPP
                    RCQVEGCGADLSGIKNYYCRHKVCFMHSKAPRVVVAGLEQRFCQQCSRFHLLPEFDQG
                    KRSCRRRLAGHNERRRRPQTPLASRYGRLAASVGEHRRFRSFTLDFSYPRVPSSVRNA
                    WPAIQPGDRISGGIQWHRNVAPHGHSSAVAGYGANTYSGQGSSSSGPPVFAGPNLPPG
                    GCLAGVGAATDSSCALSLLSTQPWDTTTHSAAASHNQAAAMSTTTSFDGNPVAPSAMA
                    GSYMAPSPWTGSRGHEGGGRSVAHQLPHEVSLDEVHPGPSHHAHFSGELELALQGNGP
                    APAPRIDPGSGSTFDQTSNTMDWSL"

ORIGIN

       1 gcaatgtaga gccacgtagg caagtcgctt gcgtggagga gagaggggag tggggaccgt
      61 tcccaaccca gcttcgtgtg accaagtttg gccacacggg ccaaacgaac ctcagcaact
     121 tttgtcagaa agaaaagaac ctccgcgaga aacaagaaag cgagagaggg agagaaagga
     181 ggctcgtcgg agtaggggcg ctcggggtat ggggctcggc ggaggctcgc tggagtaggg
     241 gccgccactg cgtggggctc gccggagtag gggcgctcgg ggagcctccc gagatccgcc
     301 gctcagcggc gccgccgtct tccgggcaga gctctcgaag ctcgccctcc tcgcgcgccg
     361 gtggcgttgg cggggcccgc gtgtggctac gcagctccgg tgctgcgcct ccaccgtcga
     421 cgacagcgcc gcttggcgct gccgccgtct tccgccgcgc cgctggacgc cgccagatct
     481 gctgctcgtc gccgcgtggg ccgctccacc cggttggagg aggagaggcg gcgccgcgct
     541 tgggctgccc caccgccgag ctctgccgcg ccgttcgccg gtgctgccga gctccgccgc
     601 gcctgccgga gcacgctgcc atggccgccc tggagaagac acgagagaat taggtggagg
     661 gtgggggaag ggtgagattt tttatattat ctatgggtcc cattataaat tttctaaacc
     721 acacttatac tgtgggtgca gtgtcattta gagttcccaa accacctatg ttgcagctgt
     781 ggtataacaa tttgctagga cgcattgcta ctgcccttgt accctgctat aagaagataa
     841 ccaatgacat ctccactcga ttttctcggc gcgcgtgtga gggtgtgagg ataattttta
     901 ttttaagtgg tttttaaggg cggagagaga gagagagaga gggcaccgca ctacttctac
     961 ttgtgtgtgt gtcgctcgct gggcttcgcc acctttccgt ctctttcctc tctcttctct
    1021 ctccccctct cctggaggag agagaggaga agaggagggg gggccgcgcc aagagccacg
    1081 cgcgctacag tctccttccc acccgcgacc gcgagcaatg gagatggcca gtggaggagg
    1141 cgccgccgcc gccgccggcg gcggagtagg cggcagcggc ggcggtggtg gtggagggga
    1201 cgagcaccgc cagctgcacg gtctcaagtt cggcaagaag atctacttcg aggacgccgc
    1261 cgcggcagca ggcggcggcg gcactggcag tggcagtggc agcgcgagcg ccgcgccgcc
    1321 gtcctcgtct tccaaggcgg cgggtggtgg acgcggcgga gggggcaaga acaaggggaa
    1381 gggcgtggcc gcggcggcgc caccgccgcc gccgccgccg ccgcggtgcc aggtggaggg
    1441 gtgcggcgcg gatctgagcg ggatcaagaa ctactactgc cgccacaagg tgtgcttcat
    1501 gcattccaag gctccccgcg tcgtcgtcgc cggcctcgag cagcgcttct gccagcagtg
    1561 cagcaggtca ctctctcact cacctcgcca ttgctgatgt caccactgct tttgctttgc
    1621 tttgcttgct ctccctcctc tttcacctat ctctcttgtt tatttgcttc ttgttcttgt
    1681 ttagtgctag tacatgtgtt gttattgttg tgccgttttg tcttttgggt tattgtgttg
    1741 ttgttactac tcgttttact ataggttttt aaggtttatg agcacggcca ccacattaga
    1801 tgcactgtca agtggtgtgt gtgggacctt tcctgctaaa acaagctgat ttcaactctc
    1861 tgaaacttcc tgcatttcat ctatttttat ctttgattgt gttgggagta ctacactagt
    1921 agtgttaata ttttgactgg tgcttatgag atttttaagt tggtaggttg atgaggaaaa
    1981 tactccttta tatggttgag tgatgtgact tgcctgtctg cctgcctgcc tgccgctttg
    2041 cataagattc ctctgtgtta gtaagagcca ctgtttattt gtactggtgc ttactctact
    2101 tagttaatta gccattagct ataaaattcc gttgatgttg caagcttagc aatggccacg
    2161 gtaagaatgg gagagagaag ttggctaaag ctgttgcttt gtagtttgta ctatatatgt
    2221 gtctttgtgt tgcaagatat gcaactccta ctatgctgtg acttgagctc aaggttttca
    2281 gttatctata gatccttact actactgagc atactaccac ttctgtatgg tagcatatgg
    2341 tagcatagtc caagttccaa cgcctcgcca gttgttcata atctatacta ccacttctgt
    2401 gcatttgtta cttttattta atagtttgtc tcattagctg acaagcatat gcctgttttg
    2461 atatctgccc ctcttgtaat agtctatgga tagcttggac tgtttgatgc tttaattttt
    2521 tactagcaac acttagggcc cctttgaaat ggaggattag caaaggaatt ttggaggatt
    2581 cattttccta aggatttttt cctatagagc cctttgattc atagaaagag gataggaaaa
    2641 cttccgtagg attgcattcc tatgatcaat tccataggaa aataagcaag aggttagacc
    2701 tcttgtgaaa ctttcctttg ttgagtgtat cttgtggtat aatcaaaggg ctcttctctc
    2761 catttcatgt gttttcaatt cctgtaggat tggaaaaaca tacaacttca attcctacgt
    2821 ttttcctatt cctatgtttt tcctatcctg cgtttcaaag gggcccttaa ggatgaaggg
    2881 aagtaagaga aacatactag agaatatgta gtagtatttc tacattccat atttgtagca
    2941 ctagcccaca aatatctttg ccttgtactt acttcatacc agttcccccc ttttcagagc
    3001 aaaccaacaa tttctgttgc cttatatatc tagtgtcttc gtactaatat atctgttcca
    3061 aaatgtacct gtccaaattc atagctagaa atagctttat ttaggacgga agtaataact
    3121 gttgttagag acttggttca gacttttggt tatgttgagg ctactatcat ttcctttacg
    3181 ggccaaatta ctacaaatga gaattcataa aaatgtcaag attttatgat tgttgtagct
    3241 ttatttagga cggaggtagt aattgttgtt agagacttgg ttcagacttt tggttacgtt
    3301 gaagctacta tcatttcctt tatggtcaaa ttactaacaa tgagtattca taaaaatgtc
    3361 aagattttat aattgagctg tgccagtgct aagtgtgtca ctatctgatg ccataatgca
    3421 tcattataaa agccagatgg accattagct tttatgtgta ggacacctgc cgtccaatta
    3481 gatggataac catctagtgt ttgtgtactg ttattttaag cccgacatct cacaactcca
    3541 tgaatgatta cagtcttcct ttcacatggt gtccttttgt tgtgttagga atagcatttt
    3601 ttatttatgg gtgtaattat gaaaggcact aggagagttg ctgctttatc ttgatgggat
    3661 ttgtagtaat accatcttta ggatgacaag aaatcttgtt ctgagttagc atgggctgcc
    3721 ttttgacctg agctacggtt tgctatgttt ggcttgcatc atgcagatct attaggataa
    3781 taagcatata aaagttgctt gcattgtgca ttgcttgttt taccttgatt catgtaggag
    3841 taatttgctc gccatgcctc gttttgcttt ctgagtcaac agccaaattt agatgatgta
    3901 ccttctgttg cttcaaaaac tcagtcactg cacagcagca gtggatagga ttcagaatca
    3961 atctatccat gattctctgt tcacataata tgacaggttc cacctgctgc ctgaatttga
    4021 ccaaggaaaa cgcagctgcc gcagacgcct tgcaggtcat aatgagcgcc ggaggaggcc
    4081 gcaaacccct ttggcatcac gctacggtcg actagctgca tctgttggtg gtatcatcag
    4141 aggctcttgt tttctttgca tcttgtgtgt ttgttggtaa ctactggttg cattcgctga
    4201 tgtgttgttt gttgcgattc ttgatccaga agagcatcgc aggttcagaa gctttacgtt
    4261 ggatttctcc tacccaaggg ttccaagcag cgtaaggaat gcatggccag caattcaacc
    4321 aggcgatcgg atctccggtg gtatccagtg gcacaggaac gtagctcctc atggtcactc
    4381 tagtgcagtg gcgggatatg gtgccaacac atacagcggc caaggtagct cttcttcagg
    4441 gccaccggtg ttcgctggcc caaatctccc tccaggtgga tgtctcgcag gggtcggtgc
    4501 cgccaccgac tcgagctgtg ctctctctct tctgtcaacc cagccatggg atactactac
    4561 ccacagtgcc gctgccagcc acaaccaggc tgcagccatg tccactacca ccagctttga
    4621 tggcaatcct gtggcaccct ccgccatggc gggtagctac atggcaccaa gcccctggac
    4681 aggttctcgg ggccatgagg gtggtggtcg gagcgtggcg caccagctac cacatgaagt
    4741 ctcacttgat gaggtgcacc ctggtcctag ccatcatgcc cacttctccg gtgagcttga
    4801 gcttgctctg caggggaacg gtccagcccc agcaccacgc atcgatcctg ggtccggcag
    4861 caccttcgac caaaccagca acacgatgga ttggtctctg tagaggctgt tccagctgcc
    4921 atcgatctgt cgtcccgcaa ggcgagtcat ggaactgaag aacctcatgc tgcctgccct
    4981 tattttgtgt tcaaattttc ctttccagta tggaaaggaa attctaaggt gactggcgat
    5041 taatctccct gtgatgaata ataatgcgcg cccttgaact caattaattg ctgtgccgca
    5101 tccatctatg taactctcca tgaattttta agtatcagtg ttaatgctgt attgtcgagg
    5161 acttctgctc gatatgttat ttctcttatg ttgttcatca tgaatctttt tctgcttatt
    5221 attctggtgc cgggttgtcc ttaccacaga agattcagtt tcggttggcg agagtaaaca
    5281 ccttccctgg ttgtgacaaa agctccaacc ttttcacttc tcggcctgta tttgatcttc
    5341 cccttctgac gctgttatac tacttttaag cctgtatgtt tccagccttc caggtgaagg
    5401 gccatactga agagaaaaca tgctttcagg gtttgatgca ttgtgtactt tacaagtgta
    5461 cttaagattt tgtacaattt atatatgtac ctgctctgct gctgagtatt gtaggaaaga
    5521 atcagttcga agggcgtgtg ttcatgtaaa gtgagaccac atgcacagcg tggatttgca
    5581 gcatgctctc tgcaccagtg gtgttctgtt gatgcctttg atgggctggc tgaggtgaga
    5641 ggaggatgat ccatgttggc agcttcttca ctctgaaaaa taaaagagaa gaaatgttca
    5701 gatttgcaga caagtggaga gcagtgatat attctacaat aaaacattac caccttgctt
    5761 ttctgtgatg atagatactc catggaattt tgcatcaagc atctcttgtt ttccagccac
    5821 tgtttgctgg gttgttgctt caatttcgtc ccaattgatt ggtcaccttt ggttgtgact
    5881 tgagagcact gagcactgaa acttttgctg tcagcaggca atgcacctca tccatgtcac
    5941 gacagaggga gagagcccac ataaatggcc aaagaggaca catacagtgg cactgatgca
    6001 gtcattgcaa cataattgac atcatgctaa acagtggtgt aaccatatgt tagctagcct
    6061 gtgatcagca aacagtgatt atggatctta atgtcacatg caagatttga cacagttgta
    6121 aaaccatcat tgcattgaag atagatccca gcaacaggtg tatgatgtat tgctagaatg
    6181 aatcaaaaat atcagtgcca tcctaaacac agtactacca acttgaacag ttatcaccgt
    6241 gattggaaaa cagaaatgta taattgcttt ggcgccatct gcttatcatt atcatatgtc
    6301 gcagatcact tgttccattg acacgactct ttttcactgt gaggagaggc accttgattt
    6361 ggacttttca agagctgtag caagggctcc tttgaagcct tctacatgga ggagcagagc
    6421 ataccatatg cagaactgta aactcttcct gaagctttcc agttccaccc ttgtagcttt
    6481 aagctgccgc aagagaatta tcatttctaa cattgagatg tgatactgaa atgtgaaagg
    6541 tgattcgcag tataggtccc aaaatatcgt ttacagcaac ttgcaaatcc tgcatgatac
    6601 agttaattca tcaaaatatt agaccattag tactacagtc tacaaatacc ccttaactga
    6661 acatgtatga taaggacaag attctgaagc tccagtgcat caggaatcca acgcagtatg
    6721 caaatcatta ctgaacaaga ttcctgcact tacagaatca tcacctgttg taacaaggac
    6781 cattctttgt tgccccagac acagcgaatt aatggtcatc ttcatttggc ccaggacatt
    6841 catttgccaa tgcttctgct gattcatact gaaaagggga caatgcgtcc aattttaaaa
    6901 gcatggaaga tgctataaaa gatcacccta tttaaaatgc agagaaaacc aaagatccaa
    6961 catgatatgg taatcacaga ttcccaacag taatgccgtc cagtaggcag taggggcatg
    7021 cacataaaca ctagtactat gtagagctga agcttatttc cagaatgaag ctgaccttgc
    7081 aaccgcaata aaggcaatag tagtgttcat cgcgcagctt agccaatata ttttgcaaat
    7141 cctgcaagaa taaacacagg tcaatctcgt cctttcagca aaatttgcag tcttgcataa
    7201 gatttctcag ataaaaaagg aagtctaga

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