荒漠旱生植物霸王角质层脂质分泌机制及其分子基础研究

The well-developed and thick surface cuticle is an effective barrier to restrict water loss and resist arid environments for xerophytes. Following its biosynthesis in epidermal cells, the cuticle, which is composed of wax and cutin, collectively termed cuticular lipids, was exported across the plasma membrane to the cell wall and further to extracellular matrix. In plants, ABCG11 gene encoding a plasma membrane-localized ATP-binding cassette transporter was suggested to play the most important and central role in the progress of cuticular lipids secretion, but plants used in these researches were mesophytes possessing thinner cuticle. In this project, based on the cloning of ABCG11 gene in our previous work, in order to reveal the function of cuticular lipid secretion mediated by ABCG11 transporter for drought adaptation of succulent xerophytes, taking the typical succulent xerophyte Zygophyllum xanthoxylum which has well-developed surface cuticle as material, the following experiments would be done: quantitative analyzing transcript abundance of ZxABCG11 under various stress conditions using real-time RT-PCR technique; analyzing chemical compositions of cuticular lipids in epidermis, observing the structure of cuticle and epidermal cells and evaluating drought tolerance of plants, after gene silencing of ZxABCG11 using RNA interference (RNAi) technique; comparing the gene expression profiles of ZxABCG11-silenced plants and wild-type Z. xanthoxylum. The results could provide theoretical basis and stress-resistant gene(s) for the genetic improvement of drought resistance in high quality forages and crops.

旱生植物表皮发达的角质层是其防止水分散失、抵御干旱环境的有效屏障。角质层组分即蜡质和角质(合称为脂质)在表皮细胞内合成后，需经质膜运输至表皮细胞外以形成角质层；ABCG11基因编码的脂质转运蛋白是参与该过程的关键基因。目前学术界对表皮角质层脂质分泌的研究均以中生植物为对象，其表皮角质层并不发达。本项目拟以表皮角质层十分发达的荒漠旱生植物霸王为材料，在前期已克隆其脂质转运蛋白基因ZxABCG11的基础上，利用qRT-PCR技术对ZxABCG11基因在不同胁迫条件下的转录丰度进行分析；采用RNA干扰技术沉默ZxABCG11基因，测定转基因植株表皮角质层各组分含量，观察其表皮角质层及表皮细胞结构并对其耐旱性进行评价；对转基因植株及野生型霸王进行表达谱比较分析。以解析角质层脂质在荒漠植物抗旱性中的作用，为优良牧草及农作物抗旱性的遗传改良提供理论依据及抗逆基因资源。

本项目取得了以下主要进展和成果：(1) 系统分析了干旱胁迫、盐处理及高温处理下ZxABCG11基因在霸王中的表达模式。(2) 成功构建了含有ZxABCG11基因、FLAG标签、安全标记基因pmi、表皮特异性启动子AtCER6及MtML1的安全高效型植物表达载体。(3) 将ZxABCG11基因转入拟南芥中并进行了干旱胁迫实验，发现转ZxABCG11拟南芥的生物量、叶片相对含水量、净光合速率、水分利用效率、叶绿素含量均高于野生型，而离体叶片失水速率、蒸腾速率、叶片相对质膜透性低于野生型，且长期干旱处理21天后，野生型植株全部死亡，而转基因株系的存活率为27%-67%，表明ZxABCG11基因的表达显著增强了拟南芥的抗旱性。(4) 通过农杆菌介导的方法将AtCER6或MtML1启动的ZxABCG11基因分别转入紫花苜蓿中，利用PCR和RT-PCR检测分别获得了2个表达量高的ZxABCG11转基因株系。(5) 对ZxABCG11转基因苜蓿进行干旱（30%田间持水量）或高温（40℃）处理，发现AtCER6驱动的AA株系的株高、地上部及根的干鲜重、离体叶片失水速率均高于野生型和MtML1驱动的MA株系，表明前者相较于后者可以更高效地驱动ZxABCG11基因的表达；通过扫描电子显微镜观察了野生型和转基因苜蓿叶表皮蜡质，发现与正常条件相比，干旱或高温胁迫下所有植株叶表皮蜡质显著增多，且转基因株系的蜡质显著多于野生型。