甘蔗TAD1(ScTAD1)的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2017.09.003

摘要/Abstract

摘要： 【目的】Tillering and Dwarf 1(TAD1)是植物株型发育的重要调控基因，该基因与腋芽的形成发育密切相关。获得甘蔗TAD1(ScTAD1)并预测其结构和功能，分析其在甘蔗不同组织部位、不同发育阶段腋芽中及在用生长素(IAA)和细胞分裂素(6-BA)处理后的蔗苗非伸长茎梢部的表达情况，以期为ScTAD1的功能分析及其在甘蔗产量分子辅助育种中的利用奠定理论基础。【方法】采用电子克隆技术并结合反转录PCR(reverse transcription PCR，RT-PCR)，cDNA末端快速扩增(rapid-amplification of cDNA ends，RACE)等技术获得ScTAD1的cDNA全长，然后利用生物信息学方法对其序列结构、功能、同源性进行分析;再使用实时荧光定量PCR(real-time fluorescent quantitative PCR，qPCR)技术对该基因在甘蔗品种ROC22不同组织部位(根、茎、叶、分蘖芽、叶鞘、生长点)、茎尖生长点和不同发育阶段腋芽(幼嫩腋芽、半大腋芽、较大腋芽、成熟休眠腋芽)及叶片分别喷施IAA和6-BA不同时间点幼苗非伸长茎梢部的表达特征进行分析。【结果】获得ScTAD1的cDNA全长(GenBank登录号为KX611166)，序列分析发现其包含1个1 560 bp的完整开放阅读框，编码519个氨基酸残基，其编码蛋白分子量为55.57 kD，理论等电点pI为9.16。保守结构域分析表明ScTAD1包含7个WD40重复序列的保守结构域;信号肽预测结果表明ScTAD1蛋白不存在信号肽，为非分泌蛋白;三级结构预测表明ScTAD1与二穗短柄草(XP_003558934.1)、玉米(XP_008650376.1)和水稻(AAN74839.1)相关同源蛋白三级结构高度相似，且与高粱假定蛋白(XP_002468612.1)亲缘关系最近。qPCR分析结果表明ScTAD1在甘蔗根、茎、叶、分蘖芽、叶鞘、生长点等不同组织部位均有表达，其中在分蘖芽中的表达量最高，其次为叶和叶鞘，根中表达较弱;不同发育阶段甘蔗腋芽中，ScTAD1在幼嫩腋芽中表达最高;叶片喷施植物激素IAA和6-BA 36 h后该基因表达开始升高，但喷施6-BA 48 h后表达又回落到未处理水平，表明这两类激素对ScTAD1表达有调控作用。【结论】成功从ROC22中获得ScTAD1的cDNA序列，该基因在甘蔗不同组织部位均有表达，其中分蘖芽中表达量最高;推测ScTAD1可能在甘蔗腋芽形成发育早期发挥作用，其表达水平受生长素和细胞分裂素调控。

关键词: 甘蔗, 腋芽, TAD1, 克隆, 表达分析

Abstract: 【Objective】The Tillering and Dwarf 1 (TAD1) gene plays an important role in regulating plant architecture, and response to the forming and developing of lateral buds. In this study, the gene named ScTAD1 was cloned from sugarcane, the structure and function of it were estimated, and the expression characteristics in different sugarcane tissues, axillary buds at different development at stages and seedlings treated by auxin and cytokinin were screened to provide a reference for function analysis of ScTAD1 and molecular assisted selection of sugarcane yield in the future. 【Method】 A series of technologies including in silico cloning, reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) were used to obtain the full-length cDNA of ScTAD1, the structure and function of this gene were analyzed and predicted using bioinformatics methods. Finally, by using the real-time fluorescent quantitative PCR (qPCR) technique, the expression pattern of this gene was detected in six different tissues (root, stem, leaf, tiller bud, leaf sheath and stem apex), stem apex and axillary buds with different size (tender axillary bud, medium axillary bud, largish axillary bud, mature dormant axillary bud) and six different time points of seedling tip with non-elongated internodes treated by IAA and 6-BA.【Result】The full length cDNA of ScTAD1 (GenBank accession number: KX611166) was obtained, which contains a 1 560 bp complete open reading frame (ORF) and encodes 519 amino acid residues with a predicted molecular weight of 55.57 kD, bioelectric point value of 9.16. Amino acid sequence analysis showed that it contains seven conserved domains named WD40 whose function is as a protein-protein or protein-DNA interaction platform. Signal peptide prediction results indicated that ScTAD1 without signal peptide and is a secreted protein. Tertiary structure prediction showed that ScTAD1 is highly similar to the homologous protein from Brachypodium distachyon (XP_003558934.1), Zea mays (XP_008650376.1) and Oryza sativa (AAN74839.1). In the phylogenetic tree, ScTAD1 has the closest evolutionary relationship with the homologous protein from Sorghum bicolor (XP_002468612.1). The qPCR analysis showed that ScTAD1 expressed in all the tested tissues, but high expression mainly occurred in tiller buds, followed by leaves, leaf sheath, and the lowest in root. At the different stages of axillary buds development, high expression of ScTAD1 gene appeared in tender buds; when the seedling was treated with IAA and 6-BA, high expression occurred in 36 h, but reduced in 48 h in 6-BA treatment, which implied that this gene can be regulated by the two plant hormones. 【Conclusion】 The ScTAD1 was successfully cloned from sugarcane, and it expressed in different sugarcane tissues with the highest expression in tiller buds. The gene ScTAD1 may play an important role in regulating the development of sugarcane axillary buds, and its expression can be regulated by the auxin and cytokinin.

Key words: sugarcane, axillary bud, TAD1 gene, cloning, expression analysis

李旭娟，字秋艳，李纯佳，刘洪博，林秀琴，徐超华，陆鑫，毛钧，刘新龙. 甘蔗TAD1(ScTAD1)的克隆与表达分析[J]. 中国农业科学, 2017, 50(9): 1571-1581.

LI XuJuan, ZI QiuYan, LI ChunJia, LIU HongBo, LIN XiuQin, XU ChaoHua, LU Xin, MAO Jun, LIU XinLong. Cloning and Expression Analysis of TAD1 (ScTAD1) in Sugarcane[J]. Scientia Agricultura Sinica, 2017, 50(9): 1571-1581.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.09.003

https://www.chinaagrisci.com/CN/Y2017/V50/I9/1571

参考文献

　　[1] XU C, WANG Y, YU Y, DUAN J, LIAO Z, XIONG G, MENG X, LIU G, QIAN Q, LI J. Degradation of MONOCULM 1 by APC/CTAD1 regulates rice tillering. Nature Communications, 2012, 3(2): 132-136.

　　[2] LI X, QIAN Q, FU Z, WANG Y, XIONG G, ZENG D, WANG X, LIU X, TENG S, HIROSHI F, YUAN M, LUOK D, HAN B, LI J. Control of tillering in rice. Nature, 2003, 422(6932): 618-621.

　　[3] SATO Y, HONG S K, TAGIRIA, KITANO H, YAMAMOTO N, NAGATO Y, MATSUOKA M. A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proceedings of the National Academy of Sciences of the USA, 1996, 93(15): 8117-8122.

　　[4] LI M, ZHANG P. The function of APC/C Cdh1 in cell cycle and beyond. Cell Division, 2009, 4(1): 1-7.

　　[5] VINARDELL J M, FEDOROVA E, CEBOLLA A, KEVEI Z, HORVATH G, KELEMEN Z, TARAYRE S, ROUDIER F, MERGAERT P, KONDOROSI A, KONDOROSI E. Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules. The Plant Cell, 2003, 15(9): 2093-2105.

　　[6] SU’UDI M, CHA J Y, JUNG M H, ERMAWATI N , HAN C D, KIM M G, WOO Y M, SON D. Potential role of the rice OsCCS52A gene in endoreduplication. Planta, 2012, 235(2): 387-397.

　　[7] 杨群. 玉米细胞周期转换蛋白ZmCCS52B调节生物量积累的分子机理[D]. 济南: 山东师范大学, 2015.

　　YANG Q. The molecular mechanism of cell cycle switch protein ZmCCS52B regulating biomass accumulation of maize[D]. Jinan: Shandong Normal University, 2015. (in Chinese)

　　[8] LIN Q, WANG D, DONG H, GU S, CHENG Z, GONG J, QIN R, JIANG L, LI G, WANG J, WU F, GUO X, ZHANG X, LEI C, WANG H, WAN J. Rice APC/CTE controls tillering by mediating the degradation of MONOCULM 1. Nature communications, 2011, 3(2): 132-136.

　　[9] LIU Y, YE W, LI B, ZHOU X, CUI Y, RUNNING MP, LIU K. CCS52A2/FZR1, a cell cycle regulator, is an essential factor for shoot apical meristem maintenance in Arabidopsis thaliana. BMC Plant Biology, 2012, 12(1): 11-12.

　　[10] BALOBAN M, VANSTRAELEN M, TARAYRE S, REUZEAU C, CUITRONE A, MERGAERT P, KONDOROSI E. Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control. New Phytologist, 2013, 198(4): 1049-1059.

　　[11] VANSTRAELEN M, BALOBAN M, DA I O, CULTRONE A, LAMMENS T, BOUDOLF V, BROWN S C, DE VEYLDER L, MERGAERT P, KONDOROSI E. APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root. Proceedings of the National Academy of Sciences of the USA, 2009, 106(106): 11806-11811.

　　[12] LARSON-RABIN Z, LI Z, MASSON P H, DAY C D. FZR2/ CCS52A1 expression is a determinant of endoreduplication and cell expansion in Arabidopsis. Plant Physiology, 2009, 149(2): 874-884.

　　[13] DANTE R A, LARKINS B A, SABELLI P A. Cell cycle control and seed development. Frontiers in Plant Science, 2014, 5(1): 493.

　　[14] HANADA T, NASHIMA K, KATO M, TAKASHINA T, IKEDA K SAKAMOTO Y, TAKAHASHI H, NAKAZONO M, OIKAWA A, SHIRATAKE K, ISUZUGAWA K. Molecular cloning and expression analysis of the WEE1 and CCS52A genes in European pear (Pyrus communis L.) and their possible roles in a giant fruit mutant. Journal of Horticultural Science & Biotechnology, 2016, 90(5): 511-517.

　　[15] MATHIEU-RIVET E, GEVAUDANT F, SICARD A, SALAR S, DO P T, MOURAS A, FERNIE A, GIBON Y, ROTHAN C, CHEVALIER C, HERNOULD M. Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endoreduplication for fruit growth in tomato. Plant Journal for Cell & Molecular Biology, 2010, 62(5): 727-741.

　　[16] 王丹. 水稻分蘖调控基因TE的功能分析和类病变突变体lms1的图位克隆[D]. 北京: 中国农业科学院, 2012.

　　WANG D. Functional analysis of a key tillering regulator TE and map-based cloning of gene LMS1 in rice (Orzya sativa L.) [D]. Beijing: Chinese Academy of Agricultural Sciences, 2012. (in Chinese)

　　[17] LIN Q, WU F, SHENG P, ZHANG Z, ZHANG X, GUO X, WANG J, CHENG Z, WANG J, WANG H, WAN J. The SnRK2-APC/CTE regulatory module mediates the antagonistic action of gibberellic acid and abscisic acid pathways. Nature communications, 2015, 6: 7981.

　　[18] LING H, WU Q, GUO J, XU L, QUE Y. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative RT-PCR. Plos one, 2014, 9(5): e97469.

　　[19] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods, 2001, 25(4): 402-408.

　　[20] HUSSIEN A, TAVAKOL E, HORNER D S, MUNOZ M, MUEHLBAUER G, ROSSINI L. Genetics of tillering in rice and barley. Plant Genome, 2014, 7(1): 93-113.

　　[21] XU C, MIN J. Structure and function of WD40 domain proteins. Protein & Cell, 2011, 2(3): 202-214.

　　[22] 许操. 水稻矮生多分蘖基因Tillering and Dwarf 1(TAD1)的克隆与功能研究[D]. 北京: 中国科学院研究生院, 2012.

　　XU C. Cloning and function research of the Tillering and Dwarf 1 gene (TAD1) in rice[D]. Beijing: Graduate University of the Chinese Academy of Sciences, 2012. (in Chinese)

　　[23] MATHIEU-RIVET E, GEVAUDANT F, SICARD A, SALAR S, DO P, MOURAS A, FERNIE A R, GIBON Y, ROTHAN C, CHEVALIER C, HERNOULD M. Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo- reduplication for fruit growth in tomato. Plant Journal for Cell & Molecular Biology, 2010, 62(5): 727-741.

　　[24] MULLER D, LEYSER O. Auxin, cytokinin and the control of shoot branching. Annals of Botany, 2011, 107(7): 1203-1212.

　　[25] HIMIZU S S, MORI H. Control of outgrowth and dormancy in axillary buds. Plant Physiology, 2001, 127(4): 1405-1413.

　　[26] 刘杨, 顾丹丹, 许俊旭, 丁艳峰, 王强盛, 李刚华, 刘正辉, 王绍华. 细胞分裂素对水稻分蘖芽生长及分蘖相关基因表达的调控. 中国农业科学, 2012, 45(1): 44-51.

　　LIU Y, GU D D, XU J X, DING Y F, WANG Q S, LI G H, LIU Z H, WANG S H. Effect of cytokinins on the growth of rice tiller buds and the expression of the genes regulating rice tillering. Scientia Agricultura Sinica, 2012, 45(1): 44-51. (in Chinese)

　　[27] 罗宝杰, 许俊旭, 丁艳锋, 李刚华, 刘正辉, 王绍华. 内源CTK和IAA平衡对水稻分蘖芽休眠与萌发的影响. 作物学报, 2014, 40(9): 1619-1628.

　　LUO B J, XU J X, DING Y F, LI G H, LIU Z H, WANG S H. Effects of endogenous hormone balance on dormancy and germination of tiller bud. Acta Agronomica Sinica, 2014, 40(9): 1619-1628. (in Chinese)

　　[28] 巩鹏涛, 李迪. 植物分枝发育的遗传控制. 分子植物育种, 2005, 3(2): 151-162.

　　GONG P T, LI D. Genetic control of plant shoot branching. Molecular Plant Breeding, 2005, 3(2): 151-162. (in Chinese)

　　[29] 刘杨, 丁艳锋, 王强盛, 李刚华, 王绍华. 激素对水稻分蘖芽生长和分蘖相关基因表达的调控效应. 植物生理学报, 2011, 47(4): 367-372.

　　LIU Y, DING Y F, WANG Q S, LI G H, WANG S H. Effect of hormones on the growth of rice tiller bud and the expression of the genes related to tiller growth. Plant Physiology Journal, 2011, 47(4): 367-372. (in Chinese)

　　[30] 刘清, 童建华, 史齐, 彭克勤, 王若仲, 蔺万煌, Mohammed Humayun Kabir, 沈革志, 萧浪涛. 一个矮秆多分蘖水稻突变体的植物激素动态特性分析. 中国农业科学, 2014, 47(13): 2519-2528.

　　LIU Q, TONG J H, SHI Q, PENG K Q, WANG R Z, LIN W H, KABIR M H, SHENG G Z, XIAO L T. Dynamic changes of phytohormones as influenced by different plant growth substances in a dwarf-multi-tiller rice mutant. Scientia Agricultura Sinica, 2014, 47(13): 2519-2528. (in Chinese)