花生AhCYP707A4基因上游转录因子分离与调控胚发育功能研究

Peanut embryo development directly impacts yield and quality. Soil calcium (Ca2+) deficiency during pegging results in poor pod filling, single kernel pods and empty pods. The underlying mechanism of this problem is also not well understood. Our previous research has shown that Ca2+ deficiency can up-regulate AhCYP707A4, leading to ABA degradation and subsequent embryo abortion. In this study, we focused on the screening and function identification of upstream transcription factors interacting with AhCYP707A4 during embryo development under Ca2+ deficiency. We screened candidate transcription factors interacting with AhCYP707A4 using a yeast one hybrid system. The expression patterns of the genes in peanut embryos under different treatments were analyzed using qRT-PCR. Also, we studied the transcriptional activity of select transcription factors which may stimulate AhCYP707A4 expression in vitro. Then, over-expression and RNAi silencing vectors with candidate transcription factors were constructed, and transformed into peanut and Nicotiana benthamiana . Phenotypic and molecular identification of transgenic plants was then performed to identify the potential functions of the candidate transcription factors in embryo development. In addition, AhCYP707A4 and select marker genes involved to ABA or Ca2+ signaling were characterized to determine if any correlation between these important genes and candidate transcription factors exists. This research sheads light on the molecular mechanisms of peanut embryo abortion, and will allow for further genetic improvement of plant yield under Ca2+ deficiency.

花生胚发育是影响产量和品质的关键事件。土壤钙不足会导致花生胚发育受阻而产生不饱满、单仁果、空荚以至烂果烂针，是生产上普遍存在的问题，迄今其分子机理未被阐明。在前期研究已经明确低钙使花生早期胚中AhCYP707A4大幅上调，引起ABA降解是导致胚败育的原因基础上，本研究拟通过酵母单杂交技术筛选AhCYP707A4上游互作转录因子，鉴定转录因子在花生胚中响应低钙胁迫的表达情况，通过离体激活AhCYP707A4表达和ABA代谢，分析其调控AhCYP707A4转录功能。构建带有候选转录因子过表达和沉默载体遗传转化花生和本氏烟草，对转基因植株进行表型与分子鉴定，同时分析转基因植株中AhCYP707A4基因、ABA信号与钙信号通路关键基因表达变化，以阐明候选转录因子功能。这对阐明花生胚发育及种子形成理论和耐缺钙花生遗传改良等都有重要的科学意义和实际价值。

花生胚发育是影响产量和品质的关键事件。土壤中钙不足可导致花生胚发育受阻而产生空荚现象，进而造成产量与品质严重下降。这一生产问题在我国南方旱地花生产区尤为突出。这也是南方花生产区只能种中小果花生品种的原因。而大果花生比中果产量高1/3以上，比小果产量高一倍以上。这也就造成我国南方花生产区产量远低于北方。然而低钙导致花生胚败育的分子机理一直未被揭示。.本研究在前期已经明确低钙导致花生AhCYP707A4基因大幅上调表达，引起ABA降解从而导致胚败育的基础上，克隆了AhCYP707A4 基因上游启动子AhCYP707A4p，构建了带有该启动子关键区域序列（含有ABA响应元件与胚发育元件）的诱饵载体pAbAi-AhCYP707A4p-bait，利用酵母单杂交技术筛选获得了与AhCYP707A4互作的转录因子环腺苷酸反应元件结合蛋白3（AhCREB3）和MYB类转录因子（AhMYB），发现了AhCREB3转录因子与胚发育关键因子AhFUS3存在互作。因此，本研究对AhCREB3、AhMYB、AhFUS3及前期获得的一个受低钙下调表达的MYB类转录因子AhGLK1进行了功能研究。利用Gateway技术构建了带有候选转录因子的超表达载体pk7WG2.0-AhCREB3、pk7WG2.0-AhMYB、pk7WG2.0-AhFUS3和pk7WG2.0-AhGLK1。利用CRISPR/Cas9技术构建了候选基因沉默载体pYLCRISPR/Cas9-pUbi-H-AhCREB3、pYLCRISPR/Cas9-pUbi-H-AhMYB、pYLCRISPR/Cas9-pUbi-H-AhFUS3，将上述载体利用花粉管导入法进行了花生遗传转化，获得了T0代转基因阳性植株。同时，利用Floral Dipping法将超表达载体转化至拟南芥中。对T2代稳定株系进行角果及胚发育情况观察。结果发现过表达AhCREB3、AhMYB与AhFUS3后，拟南芥种子都有不同程度的败育。且转基因拟南芥种子中ABA含量也显著降低。AhGLK1在拟南芥中过量表达可显著增加角果长度，并可显著降低低钙胁迫下拟南芥种子败育率。.本研究进一步揭示了AhCYP707A4调控花生低钙导致胚败育的分子机制，为花生耐低钙遗传改良奠定理论基础。