28 6 : OAP PISTILLATA(PI), AGAMOUS(AG) [2] APETALA2(AP2), MADS-box, [2-3] MADS-box MADS-box, Petunia hybrida, Antirrhina majus, Oryza stative Ze

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1 , 2011, 28(6): Journal of Zhejiang A & F University OAP3 1,2, 1, 1 2, ( ; 2., ) : MADS-box, Oncidium Milliongolds, RT-PCR (reverse transcription polymerase chain reaction) 1 MADS-box, OAP 3 (GenBank GU644447) 799 bp, 204, MADS K, Arabidopsis thaliana AP 3-like 50%~78% : OAP 3 B B C- (qrt-pcr, quantitative RT-PCR) : ; OAP : ; ; ; MADS-box ; OAP 3 ; qrt-pcr : Q781; S : A : (2011) Cloning and expression analysis of a flowering-related gene, OAP 3, in Oncidium XU Xiao-yan 1,2, TIAN Min 1, WANG Cai-xia 1, LONG Ming-hua 2 (1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang , Zhejiang, China; 2. Agricultural College, Guangxi University, Nanning , Guangxi, China) Abstract: To investigate the expression pattern of MADS-box gene during the development of the orchids flower, the floral organ identity gene OAP 3 with a MADS-box motif was cloned from an orchid plant through the RT-PCR, Oncidium Milliongolds (GenBank accession No.GU644447). Then a sequence analysis and a phylogenetic analysis were conducted along with a quantitative real-time polymerase chain reaction (qrt-pcr). The full length cdna of OAP 3 was 799 bp with an open reading frame (ORF) encoding 204 amino acids. The deduced amino acids sequence contained a typical MADS domain and a K domain, and the sequence analysis indicated that OAP 3 shared 50%-78% homology in the amino acid sequence with Arabidopsis thaliana and other species of APETALA3 homologues. The phylogenetic analysis suggested that OAP 3 belonged to the B gene, but four consensus motifs identified in the C-terminal region of the B group genes were absent in the OAP 3 gene. The qrt-pcr analysis showed that OAP 3 mrna increased with flower bud development, as well as in all four floral organs, vegetative leaves, and shoots. [Ch, 5 fig. 1 tab. 17 ref.] Key words: molecular biology; Oncidium Milliongolds ; floral initiation; MADS-box genes; OAP 3 gene; qrt-pcr [1] ABC 3, Arabidopsis thaliana APETALA1(AP1), APETALA2(AP2), APETALA3(AP3), : ; : : ( 948 ) ( ); (2009R50034) : xuxiaoyan_hope@163.com :,, tmin115@163.com

28 6 : OAP 3 901 PISTILLATA(PI), AGAMOUS(AG) [2] APETALA2(AP2), MADS-box, [2-3] MADS-box MADS-box, Petunia hybrida, Antirrhina majus, Oryza stative Zea mays [4] [5], MADS-box Orchidaceae 4,, [6],

2 28 6 : OAP PISTILLATA(PI), AGAMOUS(AG) [2] APETALA2(AP2), MADS-box, [2-3] MADS-box MADS-box, Petunia hybrida, Antirrhina majus, Oryza stative Zea mays [4] [5], MADS-box Orchidaceae 4,, [6], ABC, ABC Oncidium,, (RT-PCR,reverse transcription polymerase chain reaction) Oncidium 1 MADS-box, qrt-pcr (quantitative RT-PCR), OAP3 Oncidium Milliongolds, 5~10 mm, , 3 d 1, 6~ ( 1) -80 A. B. C. 1 Figure 1 Flower of Oncidum Milliongolds 1.2 RNA RNA RNAout 2.0 ( ) Reverse Tran scription System( Promaga ) cdna ( ncbi.nlm.nih.gov/nucleotide/), AP 3-like Primer 5.0 OF OR( 1), (PCR) AxyPrep DNA ( ) pgem-t Easy ( Promaga ), Escherichia coli DH5α( Takara ),,, AxyPrep (NCBI) BLAST, DNAMAN, CLUSTAL Neighbor-Joining (NJ), MEGA4

3 PCR Phalaenopsis hybrida ACTIN ACTINF ACTINR, 623 bp (GenBank HQ316804), Q-ACTF Q-ACTR( 1) OAP 3 Q-OAPF Q-OAPR( 1), SYBR Premix Ex Taq TM ( Takara ) (ABI) 7300 PCR 3 StepOne Microsoft Excel, ABI 2 -ΔΔC t 1 Table 1 Primers used in this study (5-3 ) OF 5 -TCATTATAGAACAACACTCGGG-3 OR 5 -TCACAGGACAAATGTAGGGG - 3 ACTINF 5 -ATTATGTTYGAGACHTTYAA -3 ACTINR 5 -ACYTTVAYYTTCATGCTGC-3 Q-OAPF 5 -TATCAGCAAGTTACCGGCTTTG-3 qrt-pcr Q-OAPR 5 -TCCCCTTTCCTCTGCCTTATC-3 qrt-pcr Q-ACTF 5 -AATGTGCCTGCTATGTATGTTGCT-3 qrt-pcr Q-ACTR 5 -GGGCATATCCTTCGTAGATTGGT-3 qrt-pcr OAP 3 : 1 MADS-box, OAP 3 ( 2), Gen- Bank GU bp, bp coding sequence (CDS), 1 203, D 9.49, (K R) 37, (D E)28, (A, I, L, F, W V)67, (N, C, Q, S, T Y) %, 13.8%, 33%, 34.97% (NCBI) BLAST DNA : * 2 OAP 3 cdna Figure 2 cdna sequence and putative amino acid sequence of OAP 3

28 6 : OAP 3 903 OAP 3, OAP 3 MADS (MADS- MEF2-like) K (K-box), 2 MADS-box 2.

4 28 6 : OAP OAP 3, OAP 3 MADS (MADS- MEF2-like) K (K-box), 2 MADS-box 2.2 OAP 3 OAP 3 MADS-box, OAP 3 B ( 3), ( 4) PeMADS5 78% MADS16 Lilium longiflorum LMADS1 62% AP 3 (50 %) DEF(50 %) AP 3, TM6 paleoaap 3 OAP 3 paleoap 3 ( 4) 3 : PI PI, SILKY1 GLO C PI MPFxFRVQPxQPNLQE [7] ; AP 3-like AP3, MADS16 LMA DS1 PI (FxFRLOPSQPNLH) [8-9] ; AP3-like euap 3 euap 3, B MADS-box AP 3-like PI, AP 3-like eu- (D(/I)ITTFALLE); AP 3-like paleoap 3 paleoap 3 (YGx- HDLRLA) [8-9] B, OAP 3 C 4, TM6 Dianthus caryophyllus CMB2 3 Figure 3 OAP 3 MADS-box B Alignment of the amino acid sequence of OAP 3 and related B group genes CLUSTAL Neighbor-Joining(NJ), MEGA4 ( 4) 4 B [10], PI, Gerbera hybrida GGLO1 PI, OsMADS4, PI

5 AP 3-like : euap 3, TM6 GDEF1 Lycopersicon esculentum TM6, paleoaap 3 B NL B OAP3 Bootstrap, 1000 : N-, S-, P-, St-, C- Figure 4 4 B Phylogenetic analysis and part expression pattern of plant B group MADS-box genes 2.3 OAP 3 OAP mm( ), 1.8 mm( ), 3.0 mm( ), 5.0 mm( ), 9.0 mm( ) 5 RNA, cdna 1, ACTIN, qrt-pcr, 5 :, OAP 3, 3.0 mm 9.0 mm OAP mm 2, OAP 3,

6 28 6 : OAP A. OAP 3 qrt-pcr B. OAP 3 qrt-pcr 5 Figure 5 3 OAP 3 Expression pattern of OAP 3 gene in Oncidium Milliongolds MADS-box, MADS-box AP 3-like OAP 3, MADS-box B B PI AP 3-like, AP 3-like 3, paleoap 3, eu- AP 3 TM6 paleoap 3 AP 3 ; euap 3 AP 3 ; TM6 AP 3 [8] OAP 3 paleoap 3 ( 3),, OAP 3 TM6, MADS TM6, 81.7%, OAP 3 TM6 GDEF1 TM ; paleoap 3, OAP 3 LMADS1 MADS ; euap3 AP 3 DEF ( 3) OAP 3, GDEF1 TM6 MADS : OAP 3 B TM6 2 : TM6 CMB2 [8] C- OAP 3 (PI, EuAP3 paleoap 3 )( 3); OAP 3 TM6 GDEF1 [9], OAP 3 B AP 3-like [9] [11],, Petunia hybrida [12-13], Lilium longiflorum [14] B ( 4), AP3, ; DEF, ; [10] GDEF1 ; SILKY1 Tulipa gesneriana, B [9,15-16] PCR, OAP3 3 mm, ( 5); OAP 3 OAP 3, B [17] 1.0 mm, 1.8 mm, 3.0 mm, 5~9 mm, 3.0 mm, OAP3 OAP 3,

7 : [1] COEN E S, MEYEROWITZ E M. The war of the whorls: genetic interactions controlling flower development [J]. Nature, 1991, 353: [2] ROUNSLEY S D, DITTA G S, YANOFSKY M F. Diverse roles for MADS-box genes in Arabidopsis development[j]. Plant Cell, 1995, 7: [3] THEIBEN G, BECKER A, DIROSA A, et al. A short history of MADS-box genes in plants [J]. Plant Mol Biol, 2000, 42: [4],,,. ppi9 [J]., 2006, 45 (3): GUO Bin, CHEN Donghong, DAI Wei, et al. Cloning and expression analysis of a Phalaenopsis flowering related gene ppi9 [J]. J Fudan Univ Nat Sci, 2006, 45 (3): [5] YU H, GOH C J. Identification and characterization of three orchid MADS-box genes of the API/AGL9 subfamily during floral transition [J]. Plant Physiol, 2000, 123: [6],,,. [M]. :, 2003: 275. [7] YANG Yingzhen, FANNICG L, JACK T. The Kdomain mediates heterodimerization of the Arabidopsis floral organ i- dentity proteins, APETALA3 and PISTILLATA [J]. Plant J, 2003, 33: [8] KRAMER E M, DORIT R L, IRISH V F. Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages [J]. Genetics, 1998, 149: [9] YU Deyue, KOTILAINEN M, P 魻 LL 魧 NEN E, et al. Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae) [J]. Plant J, 1999, 17: [10] ZAHN L M, LEEBENS-MACK J, de PAMPHILIS C W, et al. To B not to B a flower: the role of DEFICIENS and Globosa orthologs in the evolution of the angiosperms [J]. J Heredity, 2005, 96 (3): [11] JACK T, BROCKMAN L L, MEYEROWITZ E M. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens [J]. Cell, 1992, 68 (4): [12] van der KROL A R, BRUNELLE A, TSUCHIMOTO S, et al. Functional analysis of petunia floral homeotic MADS box gene PMADS1 [J]. Genes Dev, 1993, 7: [13] ANGENENT G C, FRANKEN J, BUSSCHER M, et al. The petunia MADS box gene FBP11 determines ovule identity [J]. Plant Cell, 1995, 7 (11): [14] TZENG T Y, YANG C H. A MADS box gene from lily (Lilium longiflorum) is sufficient to generate dominant negative mutation by interacting with PISTILLATA (PI) in Arabidopsis thaliana [J]. Plant Cell Physiol, 2001, 42 (10): [15] WINTER K U, BECKER A, MUNSTER T, et al. MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants [J]. Proc Natl Acad Sci USA, 1999, 96: [16] KANNO A, SAEKI H, KAMEYA T, et al. Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana) [J]. Plant Mol Biol, 2003, 52 (4): [17] MONDRAG 譫 N-PALOMINO M, THEIβEN G. MADS about the evolution of orchid flowers[j]. Trends Plant Sci, 2008, 13 (2):

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８