苹果脱水反应元件结合蛋白A4亚家族成员MdTINY的功能分析

Functions of MdTINY, a member of the apple dehydration responsive element binding-A4

摘要:

脱水反应元件结合蛋白(dehydration responsive element binding, DREB)转录因子在植物生长和发育过程中发挥重要作用，并且广泛参与各种非生物胁迫响应。DREB共含有6个亚族，TINY属于DREB-A4亚家族，拟南芥(Arabidopsis thaliana) AtTINY在植物生长与抵御逆境中起到了重要的调节作用。为了探究苹果(Malus domestica) DREB-A4亚家族的进化特征及MdTINY基因的生物学功能，本研究利用GDDH13、TAIR等数据库和Expasy、WoLF PSORT等在线软件，研究了苹果中DREB-A4亚族的生物学信息，并预测了蛋白质三级结构。苹果DREB-A4亚族含有22个基因，均含有1个保守的AP2结构域，亚细胞定位预测结果显示DREB-A4亚族蛋白主要定位于细胞核中。通过农杆菌转化法获得MdTINY的转基因愈伤组织，结合实时荧光定量PCR (quantitative real-time PCR, qRT-PCR)与花青苷含量检测，探究了MdTINY的主要生物学功能。MdTINY与AtTINY的亲缘关系更近，蛋白相似度最高，MdTINY的编码区全长为759 bp，编码252个氨基酸，启动子元件和表达模式分析表明，MdTINY基因能够响应光照和多种胁迫处理。亚细胞定位检测结果显示，MdTINY蛋白定位于细胞核中，转录自主激活活性验证实验显示，MdTINY具有自主激活活性。过表达MdTINY抑制了愈伤组织的正常生长，促进了愈伤组织的花青苷积累。以上结果表明，MdTINY负调控苹果植株生长，能够促进果实着色。本研究为苹果优质色泽品种培育提供了候选基因。

Abstract:

The dehydration responsive element binding (DREB) transcription factors play an important role in plant growth and development and are extensively involved in plant responses to abiotic stress. The DREB family contains six subfamilies, and TINY belongs to the DREB-A4 subfamily. The Arabidopsis thaliana TINY gene, AtTINY, plays a role in regulating plant growth and responses to stress. In order to investigate the evolutionary characterization of the DREB-A4 subfamily and the biological function of the MdTINY gene in apple (Malus domestica), in this study, we used the databases GDDH13 and TAIR and online tools (Expasy and WoLF PSORT) to study the biological information of the DREB-A4 subfamily in apple. In addition, the tertiary structures of the proteins were predicted. The apple DREB-A4 subfamily contained 22 genes, all of which had a conserved AP2 domain, and subcellular localization predictions showed that DREB-A4 subfamily proteins were mainly located in the nucleus. The transgenic calli of MdTINY were obtained by the Agrobacterium-mediated transformation method, and the main biological functions of MdTINY were explored by quantitative real-time PCR (qRT-PCR) combined with anthocyanin content determination. MdTINY shared the highest amino acid sequence similarity with AtTINY. The coding region of MdTINY had a full length of 759 bp, encoding 252 amino acid residues. Analysis of the promoter elements and expression patterns indicated that MdTINY was responsive to light and multiple stress conditions. MdTINY was localized in the nucleus and had transcriptional autoactivation activity. The overexpression of MdTINY in calli inhibited normal growth and promoted anthocyanoside accumulation. These results indicated that MdTINY negatively regulated apple plant growth and promoted fruit coloring, providing a candidate gene for the breeding of apple varieties with high quality of fruit color.