桂花OfAP1基因的克隆及表达分析
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摘要
AP1(APETALA1)基因在植物花分生组织及花器官形成过程中发挥着重要作用。以桂花品种‘堰虹桂’ Osmanthus fragrans ‘Yanhonggui’为材料,根据前期获得的转录组数据中AP1的序列设计引物,利用PCR技术,克隆得到约750 bp桂花AP1 cDNA序列,即Of AP1基因,其中开放阅读框长为720 bp(注册号为MH593222)。氨基酸序列比对发现,与其他物种的AP1基因的同源性高达69%～88%。荧光定量PCR结果表明:在不同组织中,桂花的Of AP1基因在花芽中的表达量显著高于其他组织,根中几乎不表达;在花芽分化过程中, Of AP1在成花转变及花芽分化初期(花瓣、花萼分化期)表达量较高,随后呈下降趋势。这说明Of AP1具有组织表达特异性,同时在桂花成花转变、花芽分化和发育中有重要作用。
Osmanthus fragrans is one of the top ten traditional flowers and a common landscaping tree in China.To better understand the function of AP1 in regulation of Osmanthus flowering, the OfAP1 gene was cloned from the Osmanthus cultivator ‘Yanhonggui', and its function was analyzed through bioinformatics method and real time PCR. Our results showed that the cDNA length of OfAP1 was 750 bp(GenBank accession No.MH593222), in which the Open Reading Frame length was 720 bp, the amino acid was 239. The OfAP1 sequence alignment revealed high homology with other species ranging from 69% to 88%. Additionally, expression profiles showed that the expression of OfAP1 in flower buds was much higher than that in other tissues(root, stem, leaf, leaf bud and flower), and the expression was barely expressed in roots. During the development of flower bud, the OfAP1 showed higher expression in S1(calyx and petal differentiation) stage than other stages. Thus, these results suggested that OfAP1 had a tissue specific expression and played an important role in flowering transformation, flower bud differentiation, and development of O. fragrans. And the results of OfAP1 can lay a foundation for further study of the molecular mechanism of O. fragrans.
Osmanthus fragrans is one of the top ten traditional flowers and a common landscaping tree in China.To better understand the function of AP1 in regulation of Osmanthus flowering, the OfAP1 gene was cloned from the Osmanthus cultivator ‘Yanhonggui', and its function was analyzed through bioinformatics method and real time PCR. Our results showed that the cDNA length of OfAP1 was 750 bp(GenBank accession No.MH593222), in which the Open Reading Frame length was 720 bp, the amino acid was 239. The OfAP1 sequence alignment revealed high homology with other species ranging from 69% to 88%. Additionally, expression profiles showed that the expression of OfAP1 in flower buds was much higher than that in other tissues(root, stem, leaf, leaf bud and flower), and the expression was barely expressed in roots. During the development of flower bud, the OfAP1 showed higher expression in S1(calyx and petal differentiation) stage than other stages. Thus, these results suggested that OfAP1 had a tissue specific expression and played an important role in flowering transformation, flower bud differentiation, and development of O. fragrans. And the results of OfAP1 can lay a foundation for further study of the molecular mechanism of O. fragrans.
引文
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[19]付建新,张超,王艺光,等.桂花组织基因表达中荧光定量PCR内参基因的筛选[J].浙江农林大学学报,2016, 33(5):727-733.FU Jianxin, ZHANG Chao, WANG Yiguang, et al. Reference gene selection for quantitative real-time polymerase chain reaction(qRT-PCR)normalization in the gene expression of sweet osmanthus tissues[J]. J Zhejiang A&F Univ, 2016, 33(5):727-733.
[20] THEISSEN G, BECKER A, DI ROSA A, et al. A short history of MADS-box genes in plants[J]. Plant Mol Biol,2000, 42(1):115-149.
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[22] LIU Chang, XI Wanyan, SHEN Lisha, et al. Regulation of floral patterning by flowering time genes[J]. Dev Cell,2009, 16(5):711-722.
[23] CHUANG Tienhsin, LI Kunhung, LI Peifang, et al. Functional analysis of an APETALA1-like MADS box gene from Eustoma grandiflorum in regulating floral transition and formation[J]. Plant Biotechnol Rep, 2018, 12(2):1-11.
[24]吴菁华,吴少华,杨超,等.建兰AP1基因的克隆、表达及其与MADS-box转录因子相互作用的分析[J].园艺学报, 2013, 40(10):1935-1942.WU Jinghua, WU Shaohua, YANG Chao, et al. Cloning and expression analysis on AP1 homologous gene from Cymbidium ensifolium and interaction analysis between AP1 and MADS-box transcription factors[J]. Acta Hortic Sin,2013, 40(10):1935-1942.
[25]吴彦庆,葛金涛,陶俊.芍药花分生组织决定基因(AP1)克隆、序列分析及其在不同发育时期花瓣中的表达变化规律[J].农业生物技术学报, 2015, 23(12):1559-1567.WU Yanqing, GE Jintao, TAO Jun. cDNA cloning, sequence analysis and tissue expression detection of APETALA1gene(AP1)in Paeonia lactiflora Pall. petals of different development stages[J]. Chin J Agric Biotechol, 2015, 23(12):1559-1567.
[26]梁芳,黄萍,袁秀云,等.菊花CmAP1基因克隆及其植物表达载体构建[J].南方农业学报, 2017, 48(6):952-959.LIANG Fang, HUANG Ping, YUAN Xiuyun, et al. Cloning of CmAP1 gene from Chrysanthemum morifolium and establishment of its plant expression vector[J]. J South Agric, 2017, 48(6):952-959.
[2] FORNARA F, de MONTAIGU A, COUPLAND G. SnapShot:control of flowering in Arabidopsis[J]. Cell, 2010, 141(3):550-550.
[3] HUANG T, B?HLENIUS H, ERIKSSON S, et al. The mRNA of the Arabidopsis gene FT moves from leaf to shoot apex and induces flowering[J]. Science, 2005, 309(5741):1694-1696.
[4] LIU Lu, ZHU Yang, SHEN Lisha, et al. Emerging insights into florigen transport[J]. Curr Opin Plant Biol, 2013, 16(5):607-613.
[5] MANDEL M A, YANOFSKY M F. A gene triggering flower formation in Arabidopsis[J]. Nature, 1995, 377(6549):522.
[6] PUTTERILL J, LAURIE R, MACKNIGHT R. It’s time to flower:the genetic control of flowering time[J]. Bioessays,2004, 26(4):363-373.
[7] MANDEL M A, GUSTAFSON-BROWN C, SAVIDGE B, et al. Molecular characterization of the Arabidopsis floral homeotic gene APETALA1[J]. Nature, 1992, 360(6401):273.
[8]戚晓利,卢孟柱.拟南芥APETALA1基因在花发育中的网络调控及其生物学功能[J].中国农学通报,2011, 27(8):103-107.QI Xiaoli, LU Mengzhu. Regulation network and biological roles of APETALA1 of Arabidopsis thaliana in flower development[J]. Chin Agric Sci Bull, 2011, 27(8):103-107.
[9] KAUFMANN K, WELLMER F, MUINO J M, et al. Orchestration of floral initiation by APETALA1[J]. Science, 2010,328(5974):85-89.
[10] IRISH V F, SUSSEX I M. Function of the APETALA1 gene during Arabidopsis floral development[J]. Plant Cell,1990, 2(8):741-753.
[11] BOWMAN J L, ALVAREZ J, WEIGEL D, et al. Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes[J]. Development, 1993, 119(3):721-743.
[12] CHEN Mingkun, LIN Ichun, YANG Changhsien. Functional analysis of three lily(Lilium longiflorum)APETALA1-like MADS box genes in regulating floral transition and formation[J]. Plant Cell Physiol, 2008, 49(5):704-717.
[13] LIU Yuexue, KONG Jin, LI Tianzhong, et al. Isolation and characterization of an APETALA1-like gene from pear(Pyrus pyrifolia)[J]. Plant Mol Biol Rep, 2013, 31(4):1031-1039.
[14] SUN Yingkun, FAN Zhengqi, LI Xinlei, et al. The APETALA1 and FRUITFUL homologs in Camellia japonica and their roles in double flower domestication[J]. Mol Breed, 2014, 33(4):821-834.
[15] LIU Yuexue, SONG Huwei, LIU Zongli, et al. Molecular characterization of loquat EjAP1 gene in relation to flowering[J]. Plant Growth Regulation, 2013, 70(3):287-296.
[16] TANG Mingyong, TAO Yanbin, XU Zengfu. Ectopic expression of Jatropha curcas APETALA1(JcAP1)caused early flowering in Arabidopsis, but not in Jatropha[J]. Peer J, 2016, 4:e1969. doi:10.7717/peerj.1969.
[17]向其柏,刘玉莲.中国桂花品种图志[M].浙江:浙江科学技术出版社, 2008:56.
[18]王英,张超,付建新,等.桂花花芽分化和花开放研究进展[J].浙江农林大学学报, 2016, 33(2):340-347.WANG Ying, ZHANG Chao, FU Jianxin, et al. Progress on flower bud differentiation and flower opening in Osmanthus fragrans[J]. J Zhejiang A&F Univ, 2016, 33(2):340-347.
[19]付建新,张超,王艺光,等.桂花组织基因表达中荧光定量PCR内参基因的筛选[J].浙江农林大学学报,2016, 33(5):727-733.FU Jianxin, ZHANG Chao, WANG Yiguang, et al. Reference gene selection for quantitative real-time polymerase chain reaction(qRT-PCR)normalization in the gene expression of sweet osmanthus tissues[J]. J Zhejiang A&F Univ, 2016, 33(5):727-733.
[20] THEISSEN G, BECKER A, DI ROSA A, et al. A short history of MADS-box genes in plants[J]. Plant Mol Biol,2000, 42(1):115-149.
[21] NG M, YANOFSKY M F. Function and evolution of the plant MADS-box gene family[J]. Nat Rev Genet, 2001, 2(3):186-195.
[22] LIU Chang, XI Wanyan, SHEN Lisha, et al. Regulation of floral patterning by flowering time genes[J]. Dev Cell,2009, 16(5):711-722.
[23] CHUANG Tienhsin, LI Kunhung, LI Peifang, et al. Functional analysis of an APETALA1-like MADS box gene from Eustoma grandiflorum in regulating floral transition and formation[J]. Plant Biotechnol Rep, 2018, 12(2):1-11.
[24]吴菁华,吴少华,杨超,等.建兰AP1基因的克隆、表达及其与MADS-box转录因子相互作用的分析[J].园艺学报, 2013, 40(10):1935-1942.WU Jinghua, WU Shaohua, YANG Chao, et al. Cloning and expression analysis on AP1 homologous gene from Cymbidium ensifolium and interaction analysis between AP1 and MADS-box transcription factors[J]. Acta Hortic Sin,2013, 40(10):1935-1942.
[25]吴彦庆,葛金涛,陶俊.芍药花分生组织决定基因(AP1)克隆、序列分析及其在不同发育时期花瓣中的表达变化规律[J].农业生物技术学报, 2015, 23(12):1559-1567.WU Yanqing, GE Jintao, TAO Jun. cDNA cloning, sequence analysis and tissue expression detection of APETALA1gene(AP1)in Paeonia lactiflora Pall. petals of different development stages[J]. Chin J Agric Biotechol, 2015, 23(12):1559-1567.
[26]梁芳,黄萍,袁秀云,等.菊花CmAP1基因克隆及其植物表达载体构建[J].南方农业学报, 2017, 48(6):952-959.LIANG Fang, HUANG Ping, YUAN Xiuyun, et al. Cloning of CmAP1 gene from Chrysanthemum morifolium and establishment of its plant expression vector[J]. J South Agric, 2017, 48(6):952-959.