缺镁柑橘叶脉木栓化机理研究

Citrus belong to evergreen subtropical fruit trees. In the major citrus production regions, citrus are cultivated mainly in acidic and strong acidic soils such as red soil, yellow soil or lateritic red soil and are usually affected by magnesium (Mg)-deficiency. Indeed, Mg-deficiency is a major factor limimting citrus yield and quality in China. Furthermore, the degree of severity is gradually increasing. Because of the severity of citrus Mg-deficiency, studies on citrus Mg-ficiency are becoming more and more. However, studies on the mechanisms for corking of Mg-deficiency citrus leaf veins are very rare. Based on our on-line retrieval, only one study compatatively observed the microscopic structure of normal leaf veins in Mg-sufficent citrus leaves and corky leaf veins in Mg-deficient citrus leaves using optical microscope. In this study, we will use ‘Xuegan’ (Citrus sinensis) as the materials and take the lead in investigating the effects of Mg-deficiency on leaf veins using optical and electron microscopes and the contents of cell wall polysaccharides and ligin and their composition in leaf veins. In addition, we will investigate the effects of Mg-deficiency on transcriptome and proteome in citrus leaf veins using RNA （Illumina/Solexa）sequencing and iTRAQ technique for the first time. The objectives of this study are to elucidate the mechanisms on corking of Mg-deficiency citrus leaf veins at microscopic structural, physiological and biochemical, and molecular biological levels, finially enhancing the tolerance of citrus plants to Mg-deficiency, and provide scientific basis for citrus cultivation of high yield and good quality.

柑橘为亚热带常绿果树，主产区土壤多为酸性或强酸性的红壤、黄壤或砖红壤，容易遭受缺镁的危害。实际上，缺镁已成为影响我国柑橘产量和品质的重要因素之一，且呈逐年加重趋势。正因为柑橘缺镁的严重性，近年来有关柑橘缺镁的研究越来越多，但很少涉及缺镁柑橘叶脉木栓化的机理。据我们的检索，目前仅有一研究利用光学显微镜对缺镁柑橘木栓化叶脉进行了观察。有鉴于此，本项目拟在前期工作的基础上，以雪柑（Citrus sinensis）为试验材料，率先研究缺镁对柑橘叶脉解剖（光学和电子显微）结构、叶脉细胞壁多糖和木质素含量及其组分的影响，并运用RNA（Illumina/Solexa）测序和iTRAQ技术研究缺镁对柑橘叶脉转录组和蛋白质组的影响，旨在从解剖、生理生化和分子生物学水平揭示缺镁柑橘叶脉木栓化的机理，最终提高柑橘的耐缺镁能力，为实现柑橘高产优质栽培提供科学依据。

本研究以雪柑（Citrus sinensis）实生苗为试验材料，率先研究缺镁（Mg）对柑橘叶脉解剖结构、叶脉非结构碳水化合物、细胞壁多糖和木质素含量，以及叶片和叶脉元素含量的影响，并运用RNA-Seq测序和Label-free技术研究了缺Mg对柑橘叶脉转录组和蛋白质组的影响，旨在从解剖、生理生化和分子生物学水平揭示缺Mg柑橘叶脉木质化的机理。主要结果如下：.a) 缺Mg症状首先出现在基部老叶，起初表现为脉间黄化，黄化从叶缘开始，然后往叶尖扩展，仅在叶基部留下一个倒“V”的绿色区域。大量缺Mg处理的下部叶和少量中部叶，其叶中脉和主（侧）脉出现像缺硼（B）一样的叶脉肿大和爆裂。.b) 缺Mg并不显著影响柑橘叶脉B含量，缺Mg叶脉肿大和木质化并是缺Mg导致的缺B所致。.c) 叶脉解剖结构分析表明，缺Mg可能引起韧皮部紊乱，从而妨碍韧皮部的运输，导致叶绿体中碳水化合物的积累和次生细胞壁木质化；缺Mg引起的形成层衍生的次生木质部和韧皮部的过度增加可能是导致叶脉肿大和劈裂的原因。.d) 缺Mg增加下部叶叶脉木质素含量，减少下部叶叶脉纤维素含量；缺Mg减少下部叶叶脉总的可溶性（葡萄糖 + 果糖 + 蔗糖）、淀粉和总的非结构性碳水化合物（葡萄糖 + 果糖 + 蔗糖 + 淀粉）含量，但并不显著影响其葡萄糖、果糖和蔗糖含量。.e) 缺Mg诱导的下部叶叶脉木质化含量的增加可能与以下几个方面有关：(a) 缺Mg引起的下部叶叶脉纤维素和淀粉积累减少可能促进了木质素的合成；(b)很多与木质素生合成有关的基因（蛋白质）其表达（丰富性）在缺Mg下部叶叶脉中均显著增加，尤其是参与苯丙酸生物合成的基因与蛋白质增加更为明显。.f) 生理、转录组和蛋白组分析表明，缺Mg对柑橘下部叶叶脉的影响远大于对上部叶叶脉的影响，缺Mg加大了上下部叶叶脉之间的差异。