【作者】 倪志勇；

【导师】 张辉；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 大豆是我国主要的油料经济作物，由于我国大豆产量及含油量低、易受干旱和盐碱等非生物胁迫影响等特点，我国每年需要进口六千万吨以上才能满足需求。因此培育高产、优质、抗逆大豆新品种是我国大豆产业亟待解决的重大问题。研究表明，植物miRNA在应答非生物胁迫中具有重要的作用。因此，大豆抗逆miRNA的功能研究对阐明其抗逆机理及其调控网络、培育高产、优质、抗逆大豆新品种具有较高的科学意义及应用价值。本研究分析了大豆gma-miR169c基因及其靶位点GmNFYA3和gma-miR394a基因在干旱胁迫中的生物学功能，旨在解释大豆抗旱抗逆机理及其调控网络，为大豆的抗逆研究及新品种培育提理论基础。1、本研究克隆了一个大豆gma-miR169c基因的GmNFYA3靶位点，它编码NF-Y复合物中的NF-YA亚基。Real time RT-PCR分析表明GmNFYA3受脱落酸（ABA）和非生物胁迫，如PEG，NaCl和冷诱导。亚细胞定位分析表明GmNFYA3可能在细胞核中激活它特异的靶位点。GUS组织化学染色分析表明在转基因拟南芥的不同组织中GmNFYA3启动子驱动的GUS基因均有表达。烟草共注射和5’ RACE实验表明miR169在体内直接切割GmNFYA3mRNA。在拟南芥中，过表达GmNFYA3减少叶片水分丧失且增强转基因拟南芥的干旱忍耐能力。另外，转基因拟南芥表现出增加对高盐和外源ABA的敏感性。而且，在正常条件下，相比野生型对照，过表达GmNFYA3株系中ABA代谢（ABA1，ABA2），ABA信号（ABI1，ABI2）和胁迫应答基因包括RD29A和CBF3的转录水平显著提高。这些结果表明，GmNFYA3靶位点是一个干旱胁迫忍耐的正调控子。2、本研究分析了大豆gma-MIR394a基因的功能，特别是它在干旱胁迫忍耐中的作用。表达模式分析表明在大豆各种组织中gma-MIR394a的表达不同且在叶片中gma-MIR394a的表达受干旱、高盐、低温和ABA处理诱导。预测了一个gma-MIR394a靶mRNAGlyma08g11030，并通过修饰的5’ RLM-RACE实验进行了验证。过表达gma-MIR394a减少叶片水分丧失且增强转基因拟南芥的干旱忍耐能力。而且，在拟南芥中过表达gma-MIR394a减少一个包含miR394a互补位点的拟南芥F-box基因（At1g27340）的转录水平。这些结果表明，gma-MIR394a基因在干旱胁迫忍耐中具有正调控功能。更多还原

【Abstract】 Soybean (Glycine max L. Merr.) is China’s leading economic oilseed crop. Due to the low yieldand oil content of soybean in China, and soybean is vulnerable affected by a wide range of abioticstresses, such as drought and salinity, China’s annual needs more than sixty million tons of soybeanimport to meet demand. Therefore, breeding for high yielding, high quality, stress tolerance soybeanvariety is needed to solve major problems in China’s soybean industry. Recent evidence indicates thatplant microRNAs have an important function in adaptive responses to abiotic stresses. Thus, toelucidate the molecular adaptation mechanisms of stress and stress regulatory networks, breeding forhigh yielding, high quality, stress tolerance soybean variety, functional studies of stress-responsivemiRNA in soybean has high scientific significance and application value. In this paper, we described indetail the biological functions of soybean gma-miR169c and its target gene GmNFYA3, andgma-miR394a in drought stresses. This research will explain the mechanism and regulation network ofsoybean drought tolerance, provides the theoretical basis for soybean stress tolerance research andcultivation of new varieties. The main results were listed as follows:1. In this study, we identified and characterized a gene, GmNFYA3, which encodes the NF-YAsubunit of the NF-Y complex in soybeans. Real time RT-PCR analysis indicated that GmNFYA3wasinduced by abscisic acid (ABA) and abiotic stresses, such as PEG, and cold. Subcellular localizationanalysis suggested that GmNFYA3may activate its specific targets in the nucleus. Histochemicalβ-glucuronidase (GUS) staining revealed that the expression of the GUS gene driven by the GmNFYA3promoter occurred in various transgenic Arabidopsis tissues. Coexpression in Nicotiana benthamianaand5’ RACE assays indicated that miR169directs GmNFYA3mRNA cleavage in vivo. Overexpressionof GmNFYA3resulted in Arabidopsis with reduced leaf water loss and enhanced drought tolerance. Inaddition, the transgenic Arabidopsis exhibited increased sensitivity to high salinity and exogenous ABA.Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI1, ABI2) andstress-responsive genes, including RD29A and CBF3, were generally higher in GmNFYA3plants than inwild-type controls under normal conditions. These results suggest that the GmNFYA3gene functions inpositive modulation of drought stress tolerance and has potential applications in molecular breeding toenhance drought tolerance in crops.2. In this study, a soybean gma-MIR394a gene was functionally characterized, especially withregard to its role in drought stress resistance. Expression analysis revealed that gma-MIR394a wasexpressed differentially in various soybean tissues and was induced by drought, high salinity, lowtemperature stress, and abscisic acid treatment in leaves. One target gene of gma-miR394a,Glyma08g11030, was predicted and verified using a modified5’ RLM-RACE (RNA ligase-mediatedrapid amplification of5’ cDNA ends) assay. Overexpression of gma-MIR394a resulted in plants withlowered leaf water loss and enhanced drought tolerance. Furthermore, overexpression of gma-MIR394a in Arabidopsis reduced the transcript of an F-box gene (At1g27340) containing a miR394complementary target site. These results suggest that the gma-MIR394a gene functions in positivemodulation of drought stress tolerance and has potential applications in molecular breeding to enhancedrought tolerance in crops.更多还原