花生油质蛋白基因调控耐盐性的分子机制解析

Oleosins, a kind of low molecular weight basic proteins studding in the surface of oil bodies, have the function of maintaining the oil body’s stability and also play an important role in plant stress resistance. By means of the peanut transcriptome and digital gene expression profiles conducted by this research group and in combination with the RT-qPCR analysis, four oleosin genes whose transcription levels were differentially expressed between the salinity-tolerant mutants and the mutagenic parents and were significantly responsive to various stress treatments were identified. Further study showed that the expression level of these oleosin genes were in agreement with the development level and oil content of the seeds. Therefore, it is deduced that the oleosin genes may be correlated with the salinity tolerance and oil synthesis of peanut. To identify the function of oleosin genes, oleosin genes whose transcription levels were significantly up-regulated after salinity treatment will be transformed into peanut to analyze the salinity tolerance, oil content and oil body size of transgenic peanut and its control in our current study. Its promoter will be cloned, and a serious of constructs including the complete promoter sequence and serial deletions in the promoter region will be constructed and transformed to analyze the important salt stress responsive elements and seed specific expression elements. Subsequently, some transcription factors will be screened out by yeast one hybrid system and the function of them will be verificated to elucidate the regulatory mechanism. The research will provide not only new genes and promoters with intellectual property rights for peanut breeding with salinity tolerance and high oil content, but also a theoretical basis for elucidating the regulatory network of salinity tolerance and oil synthesis.

油质蛋白是一类镶嵌在油体表面的碱性小分子蛋白，可维持油体的稳定性，在植物抵抗逆境胁迫中也发挥着重要作用。本课题组利用构建的花生转录组和表达谱，结合荧光定量PCR分析，筛选到4个在耐盐突变体和诱变亲本中差异表达，且对多种胁迫处理有明显响应的油质蛋白基因。进一步研究发现这些基因的表达量和种子的发育以及含油量趋势一致。我们推测油质蛋白基因可能与花生的耐盐性和油脂合成密切相关。本项目拟在此基础上，将盐胁迫处理后显著上调的油质蛋白基因导入花生，分析转基因花生的耐盐性、种子的含油量和油体的大小，确认该基因的功能；克隆其启动子，构建全长及缺失载体并进行遗传转化，验证该启动子上的盐胁迫响应元件和种子特异表达元件；通过酵母单杂交筛选与这些元件结合的转录因子并进行功能验证，解析其调控机理。本研究不仅为花生耐盐、高油育种提供具有自主知识产权的新基因和启动子，也为揭示花生耐盐和油脂合成的调控网络提供理论依据。

油质蛋白是一类镶嵌在油体表面的碱性小分子蛋白，可维持油体的稳定性，在植物抵抗逆境胁迫中也发挥着重要作用，但油质蛋白基因与花生耐盐性的关系未见报道，其分子机理更不清晰。本研究从花生耐盐突变体和诱变亲本中筛选到差异表达的油质蛋白基因，本试验对其中1个基因AhOLE2进行了克隆和遗传转化。在花生中过表达AhOLE2基因后，转基因花生的耐盐性显著提高。在盐胁迫处理下，与未转基因对照相比，过表达AhOLE2的转基因花生植株具有较高的相对含水量、叶绿素含量、光系统II的最大光化学效率和脯氨酸含量，而H2O2含量、O2-含量、MDA含量和相对电导率显著降低。转基因花生种子的含油量比未转基因对照增加约3个百分点。 . 用pAhOLE2启动子系列截短片段驱动GUS基因，转化拟南芥后，发现pAhOLE2启动子受ABA、NaCl的诱导，含有257bp的截短片段仍具有驱动活性。通过关键元件的突变分析，发现GBL元件（ACGTG）和RYL元件（CATG）可能是影响启动子响应盐胁迫和调控种子发育的关键元件。酵母单杂交试验和双荧光素酶瞬时表达试验表明：bZIP类转录因子AhABI5和B3转录因子AhFUS3均能结合pAhOLE2启动子并激活pAhOLE2驱动的基因表达。. 在花生中过表达AhFUS3基因，转基因花生的耐盐性显著提高。在盐胁迫处理下，与未转基因对照相比，过表达AhFUS3的转基因花生植株具有较高的光系统II的最大光化学效率和脯氨酸含量，而H2O2含量、O2-含量、MDA含量和相对电导率显著降低。同时发现过表达AhFUS3基因后，转基因花生种子的含油量比未转基因对照增加5个百分点以上。. 本研究不仅为花生耐盐、高油育种提供具有自主知识产权的新基因和启动子，也为揭示花生耐盐和油脂合成的调控网络提供理论依据。