甘蔗TAD1(ScTAD1)的克隆与表达分析

Cloning and Expression Analysis of TAD1(ScTAD1) in Sugarcane

【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),c DNA末端快速扩增(rapid-amplification of c DNA ends,RACE)等技术获得ScTAD1的c DNA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,q PCR)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c DNA全长(Gen Bank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 k D,理论等电点p I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。q PCR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c DNA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。

[ Objective ] The Tillering and Dwarf I （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.