子莲响应连作障碍的转录组和蛋白质组整合研究

Continuous monoculture problem seriously affects the sustainable development of property in Nelumbo nucifera. In our previous research, the physiological damage was illustrated in N. nucifera, causing by continuous monoculture. However, the molecular mechanism of continuous monoculture is still unvailed in N. nucifera. It mightbe uncover the mechanism of continuous monoculture, furthor studying the key genes and proteins in response to continuous monoculture. In this project, the variaty “Taikonglian no.36” will be planted as experimental material. The continous monoculture of N. nucifera is studied as treatments, using the normal growth of planta as control. Both total RNAs and proteins will be exacted from the roots in treatment group as well as control one. The future study will be focused on three aspects as follow. Firstly, using Solexa and iTRAQ sequencing respectively, the genes and proteins in response to continuous monoculture will be identified by comparison the expression profiles of treatment and control group. Correlation analysis of genes and proteins will be performed in order to achieve the candidate responding factors. Secondly, in combination with study of the symptoms of continuous monoculture, the spatial and temporal expression patterns of these candidate responding factors will be examined using qPCR and Western blotting technologies, in order to gain the key ones related to continuous monoculture. Finally, the homology model and bioinformatic will be performed to understand the 3D-structural of key proteins, and the recombinant proteins will be expressed and purified for the aim of studying their characterics. This project is sought to illustrate the signal transduction by replanting disease in N. nucifera, and provide theoretical basis for resoving continuous monoculture problem by the way of reforming the key molecular or regulationg continuous monoculture.

连作导致子莲结实率下降，腐败病发病率上升。目前，子莲连作障碍的分子机制尚不明确。本实验室前期研究了子莲连作的胁迫效应，以其响应连作胁迫的应答机制为切入点，有可能进一步揭示连作障碍的机理。本项目拟以子莲品种“太空莲36号”为实验材料，连作子莲为处理，新植子莲为对照，在营养生长期收集子莲根，进行以下研究：①采用Solexa核酸和iTRAQ蛋白测序技术，挖掘子莲响应连作胁迫的应答基因和蛋白，对其进行关联性分析获得候选应答因子；②通过qPCR和Western blot技术，分析候选应答因子在子莲不同组织和生长时期的表达，筛选时空表达模式与连作症状相吻合的关键应答因子；③采用生物信息学方法，结合体外表达纯化技术，研究关键应答蛋白的高级结构和生理功能。通过“转录组-蛋白质组-连作症状”整合研究，初步阐明子莲响应连作胁迫的感知、传递和应答途径，为从分子水平“纠错”，开发子莲耐连作育种技术提供理论依据。

子莲连作障碍的分子机制尚不明确。我们采用Solexa核酸和iTRAQ蛋白测序技术，挖掘子莲响应连作胁迫的应答基因和蛋白，对其进行关联性分析获得候选应答因子，并对关键基因的功能进行了研究。通过“转录组-蛋白质组-连作症状”整合研究，阐明子莲响应连作胁迫的感知、传递和应答途径，为从分子水平“纠错”，开发子莲耐连作育种技术提供理论依据。取得的研究成果如下：.一、通过测序分析共获得了9,810个差异表达基因和975个差异表达蛋白，关联性分析共获得438个差异表达转录本和蛋白质。对关联上的转录本和蛋白进行GO注释，COG分类及KEGG数据库生物合成途径分析，对差异表达的蛋白和基因进行聚类分析。研究关键基因在子莲藕鞭，根和叶中的差异性表达。关联分析表明连作可以诱导子莲苯丙素合成途径、类黄酮合成通路和MAPK信号通路。推测子莲连作障碍通过调控子莲DNA甲基化水平，影响木质素及类黄酮代谢平衡，抑制抗氧化系统酶的活力及表达，增强活性氧离子及乙烯的合成。.二、我们克隆了两个脱水素NnDHN1和NnDHN2基因，两者具有不同的等电点、酶活及功能结构。系统进化研究表明NnDHN1和NnDHN2蛋白分别属于YnSKn–型及SKn–型DHN。两个基因的表达模式存在差异。ABA和IAA均可以诱导叶片NnDHN2基因表达上调。然而NnDHN1基因在ABA的处理下表达量升高，IAA处理下NnDHN1基因表达量变化较小。NnDHN1和NnDHN2基因在响应外界环境胁迫方面具有独特的作用，但功能具有部分重叠。.三、类黄酮合成酶（FLS）是苯丙氨酸代谢的关键酶。我们克隆了4个NnFLS基因 。NnFLS蛋白具有不同的等电点、酶活及功能结构。4个NnFLS基因都含有3个外显子和2个内含子，除了NnFLS4基因第一个内含子剪切方式为GC-AG，其它内含子剪切方式都符合GT-AG。进化树分析表明NnFLS2与双子叶植物FLS聚类在一起，其它3个基因与单子叶植物FLS基因进化关系更近。NnFLS2，NnFLS3和NnFLS4在胚芽里的表达量最高，在幼叶里的表达居中，其次为根和茎。而NnFLS1在胚芽里的表达量最高，根里表达量居中，幼叶和茎里表达量最低。冷胁迫和机械损伤均可以诱导NnFLS基因的表达。