花生种子特异性高表达启动子的全基因组筛选、克隆及功能鉴定

Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world. With the development of biotechnology, researchers have been attempting to improve the seed quality of peanut through genetic engineering. This will need to express gene(s) in a seed-specific manner. Therefore, much effort has been focused on the isolation and identification of seed-specific promoter in peanut. However, this only resulted in very few such promoters being identified currently. The peanut genome sequence has been completed and the data are now available online. This, coupled with the decreasing for the cost of RNA-Seq technology will no doubt accelerate the isolation of further seed-specific promoters in peanuts in the future. Indeed, we recently isolated two promoters for genes encoding the peanut seed storage protein Ara h2.02 and Ara h6. Preliminary study carried out in Arabidopsis to express the reporter protein GUS (β-glucuronidase gene) under the control of one of the promoters showed seed-specific staining of GUS. This indicates that this promoter can indeed direct the seed-specific expression of protein. In this study we will try to isolate more seed-specific promoters by identifying genes that are preferentially expressed in seeds. This will be done by sequencing the transcriptomes of RNAs isolated from different tissues of peanut. The promoters of the seed-specific genes will be isolated from the genome DNA. And their functions will be characterized in transgenic Arabidopsis by the GUS expression and staining assays. The core functional elements of some of the seed-specific promoters that show high activity will be identified through a series deletion of the promoter sequences. The activities of the full length and the individual fragments of these promoters will be compared with the constitutive CaMV 35S promoter and the seed-specific Napin promoter from B. napus. We aim to select several novel promoters that can drive expression of transgenes specifically in seeds with high efficiency. This will provide tools for transgenic improvement of seed quality as well as to explore the mechanism of seed development of peanut.

花生是世界重要的油料作物，随着生物技术的发展，利用基因工程对花生种子进行品质改良的研究越来越多。作为品质改良重要的分子工具，花生种子特异启动子可驱动基因只在种子中表达，逐渐受到研究者的重视。我们也克隆了花生两个贮藏蛋白基因的启动子，其中一个已初步鉴定具有种子特异性。然而，可供选择使用的花生种子特异启动子还是比较少，尤其是高效表达、片段较小的种子特异启动子。目前，转录组测序成本逐渐降低，花生基因组测序已经完成，我们拟对花生不同组织的转录组进行测序，全基因组规模筛选花生种子特异高表达基因，构建其启动子驱动报告基因GUS的植物表达载体，在转基因拟南芥中鉴定其功能；利用片段缺失试验，鉴定保留种子表达特异性的较小片段；通过与35S和Napin启动子活性进行比较，最终获得花生种子特异性高表达启动子。本研究将极大丰富花生种子特异启动子资源，对花生种子品质改良以及研究花生种子生长发育的机制等具有重要意义。

花生是世界重要的油料作物。随着生物技术的发展，利用基因工程对花生种子进行品质改良的研究越来越多。作为品质改良重要的分子工具，花生种子特异启动子可驱动基因只在种子中表达，逐渐受到研究者的重视。本项目通过比较花生种子和非种子组织的转录组数据，挖掘到337个可能的花生种子特异表达基因（Seed specific candidate genes, SSCG）。我们将其中表达量前108的基因进行了鉴定，54个基因与种子发育或种子贮藏蛋白等有关。基于RNA-seq数据和qRT-PCR分析发现，大部分基因在种子中特异表达或优势表达。RY REPEAT和GCN4等顺式作用元件通常存在于种子或胚特异表达启动子，我们分析发现92个SSCG的启动子序列具有RY REPEAT元件，33个启动子序列具有GCN4元件。本研究累计克隆获得了表达量较高的28个SSCG的启动子片段，分别构建了驱动GUS报告基因的植物表达载体。转基因拟南芥实验发现：AHSSP1、AHSSP2、AHSSP29等启动子是种子特异表达启动子，他们可以驱动GUS报告基因在转基因拟南芥种子、萌发的子叶甚至长出真叶后的子叶中表达;而在转基因拟南芥莲座叶、茎生叶等非种子组织中不表达。以上结果表明：我们全基因组筛选花生种子特异表达基因的方法可靠；鉴定的SSCG的启动子可以用于作物种子品质改良或以种子作为生物反应器的研究。本项目极大丰富了花生种子特异启动子资源，具有重要理论价值和应用价值。