中国农业科学2017，Vol.50Issue(9)：1571-1581,11.DOI:10.3864/j.issn.0578-1752.2017.09.003
甘蔗TAD1(ScTAD1)的克隆与表达分析
Cloning and Expression Analysis of TAD1 (ScTAD1) in Sugarcane
摘要
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 1560 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.关键词
甘蔗/腋芽/TAD1/克隆/表达分析Key words
sugarcane/axillary bud/TAD1 gene/cloning/expression analysis引用本文复制引用
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):1571-1581,11.基金项目
国家自然科学基金(31360359,31601362)、云南省基础研究计划青年项目(2015FD063)、云南省中青年学术技术带头人后备人才(2014HB038) （31360359,31601362）
