火龙果HubHLH1转录因子应答逆境胁迫的功能解析

Basic heliex-loop-helix proteins (bHLHs) are found throughout the eukaryotic kingdom, and constitute one of the largest families of plant transcription factors. bHLHs are necessary for plant normal growth and development, and play important roles in abiotic-stress responses. However, we know little about their origins, structures, and functions due to the large quantities and complexity of plant bHLH family. In our previous study, suppression subtractive hybridization (SSH) and cDNA microarray approaches were combined to identify the potential important or novel genes involved in the dragon fruit responses to drought stress. A transcription factor bHLH122-like homolog (HubHLH1) was found to be the most abundant transcripts. These results indicated that HubHLH1 are functional in dragon fruit response to abiotic stresses. Here the functions of HubHLH1 in stress response were further investigated. The study is mainly presented as follows: the expression profiles of HubHLH1 will be analyzed by droplet digital PCR analysis; Subcellular localization will be analyzed by transient expression vector; in vitro assay of recombinant HubHLH1 protein will be detected; the over-expression and RNAi manipulation of HubHLH1 will be performed using Arabidopsis thaliana as explants. Then the phenotypic characteristics and gene expression analysis of transgenic lines will be carried out to verify the HubHLH1’s functions. Net fluxes of H+ will be measured noninvasively using scanning ion-selective electrode technique; The JIP test will be exploited to assess primary photochemical reaction and photosynthetic apparatus of transgenic plant. The study will provide a direct evidence for the important applications of HubHLH1 in dragon fruit improvement and germplasm innovation.

bHLH是真核生物中存在最广泛的一大类转录因子，它不仅与植物的正常生长和发育有关，同时也参与植物适应多种逆境胁迫的反应过程。火龙果具有很强抗旱性，我们在前期研究中得到一个干旱胁迫下高丰度表达的火龙果bHLH转录因子（Hu bHLH1），说明其可能在火龙果应答干旱胁迫中发挥着某些重要作用，迄今尚无火龙果bHLH转录因子调控抗逆性的报道。本项目拟深入探讨HubHLH1转录因子在火龙果应答干旱等逆境胁迫过程中的功能，包括原核表达检测HubHLH1重组蛋白的体外活性；构建HubHLH1基因超量表达和干涉表达载体，转化拟南芥、烟草，观察转基因株系表型，利用数字定量PCR技术检测基因表达；利用非损伤微测技术测定转基因株系的H+ flux；应用叶绿素荧光诱导动力学曲线分析转基因株系光合机构的结构和状态等变化。项目的开展，将揭示HubHLH1参与火龙果抗逆的机制，也将为深刻理解bHLH的功能提供新信息。

bHLH转录因子参与植物对逆境胁迫的反应过程。前期研究从火龙果抗旱基因芯片中筛选发现一个与bHLH转录因子高度同源的EST在干旱下高丰度表达，表明bHLH转录因子可能在火龙果应答干旱胁迫的过程中起重要作用。本项目对该转录因子进行了全长克隆并进行了功能验证，主要研究结果如下：检测了该基因在干旱胁迫下的表达情况，发现干旱诱导其高表达，并在15 d时表达量达到最大；利用RACE技术克隆了该基因，共获得2,343 bp全长序列，其中开放阅读框1,221 bp，编码406个氨基酸，命名为HubHLH1，基因登录号KU976397.1；生物信息学分析，该基因分子量45.19 kD，理论等电点pI为8.65，并在340-389 aa有一个HLH保守结构域，属亲水性不稳定蛋白，存在26个丝氨酸（Ser）和2个苏氨酸（Thr）磷酸化位点，系统进化树表明HubHLH1结构域在进化过程中保守程度较高，与双子叶被子植物的亲缘关系较近；亚细胞定位结果表明HubHLH1存在于细胞核上，具有核定位信号；通过荧光定量PCR分析该基因在非生物胁迫下的表达情况，结果发现，30 d干旱时期内HubHLH1表达上调，整体呈“N”型表达趋势，高温、低温及SA胁迫下，该基因表达量均呈上升趋势，在ABA处理下，其表达量变化不显著，在H2O2处理下，表达量显著下调；采用农杆菌介导叶盘转化法将HubHLH1基因转化烟草，获得10株转基因株系；对转基因植株实施干旱胁迫后，发现转基因植株脯氨酸和可溶性糖含量显著提高，丙二醛含量显著下降，H2O2活性下降，POD活性显著提高。项目的研究丰富了bHLH转录因子的生物学功能，揭示了HubHLH1参与火龙果抗逆的机制，为火龙果品种改良提供参考依据。