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【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】 Tillering and Dwarf 1 (TAD1) is an important regulatory gene of plant plant type, which is closely related to the formation and development of axillary buds. Sugarcane TAD1 (ScTAD1) was obtained and its structure and function were predicted. Its sugarcane seedlings were planted in axillary buds of different tissues and stages of sugarcane, and after treatment with IAA and 6-BA Long stem tip expression, in order to provide a theoretical basis for the functional analysis of ScTAD1 and its utilization in molecular-assisted breeding of sugar cane. 【Method】 The full length cDNA of ScTAD1 was obtained by using electronic cloning technology combined with reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) Then the sequence structure, function and homology were analyzed by using bioinformatics method. Then the gene was analyzed in different tissue parts of ROC22 (root, shoot, Stems, leaves, tiller buds, leaf sheaths and growth points), shoot tip growth and axillary buds at different developmental stages (young axillary buds, axillary buds with large buds, axillary buds with large axillary buds and mature buds) and leaves were sprayed with IAA and 6-BA At the time point, the expression characteristics of non-elongated shoot tips of seedlings were analyzed. 【Result】 The full-length cDNA of ScTAD1 was obtained (GenBank accession number: KX611166). Sequence analysis revealed that it contained a complete open reading frame of 1 560 bp, encoding 519 amino acid residues, encoding a protein with a molecular weight of 55.57 kD , The theoretical isoelectric point p I is 9.16. Conserved domain analysis showed that ScTAD1 contains seven conserved domains of WD40 repeats; signal peptide prediction results show that there is no signal peptide in ScTAD1 protein, which is non-secreted protein; tertiary structure prediction shows that ScTAD1 and Brachymystax sp. (XP_003558934.1 ), The tertiary structure of homologous proteins of maize (XP_008650376.1) and rice (AAN74839.1) are highly similar and have the closest genetic relationship with the putative protein of Sorghum (XP_002468612.1). q PCR analysis showed that ScTAD1 was expressed in different tissues such as root, stem, leaf, tiller bud, leaf sheath and growth point of sugarcane, among which the highest expression was in tiller bud, followed by leaf and sheath, and the expression in root was weak ; ScTAD1 was the highest expression in axillary buds of young sugarcane at different developmental stages; the expression of ScTAD1 was up-regulated after IAA and 6-BA were sprayed for 36 h in leaves, but down-regulated after 6-BA 48 h Untreated levels indicate that these two hormones regulate the expression of ScTAD1. 【Conclusion】 The c DNA sequence of ScTAD1 was successfully obtained from ROC22. The gene was expressed in different tissues of sugarcane, and the highest expression was found in tiller buds. It was speculated that ScTAD1 might play an important role in the early development of sugarcane axillary buds, Su and cytokinin regulation.