新疆北部早春短命植物独行菜种子低温萌发停滞的调控机制

Lepidium (Lepidium apetalum Willd) living in northern Xinjiang is a typical spring ephemeral plants , which plays an important role in the plants distribution of Xinjiang. And Lepidium can germinate at low temperature in early spring. However, the tolerance mechanism of germination in cold was rarely reported. It was found that there will be a period of stagnation when the Lepidium seed germination to a certain stage if they are still in low temperature. And the stagnation of germination at cold can be broken with higher temperature than 8℃ for a short time. This project will study how break the stagnant of low temperature germination of Lepidium and why the high temperature can break the stagnant. First of all, it will be used two-dimensional electrophoresis and Solexa techniques to screen expression genes induced by higher temperature than 8℃ respectively from the protein and mRNA level. Second, the function of these genes will be to predict through the comparison of functional genomics and comparative proteomics. Third, the expression changes of these genes before and after high temperature treatment were studied by real-time RT-PCR to expect to know the correlation between the expression level and their break germination stagnant. Fourth, over-expression or silencing of the gene in Arabidopsis will be realized with the transgenic technology and RNA interference technology to analyze the effect of these genes on breaking germination stagnant of Lepidium seed germination, and elucidate the regulation mechanisms in molecular level. This study would also provide the basis for revealing the molecular mechanisms of low temperature germination for other early spring ephemeral plants in early spring.

分布于新疆北部的独行菜(Lepidium apetalum Willd)属典型的早春短命植物，在本地植物分布区系中具有重要的地位，能在早春适应低温进行萌发。前期研究发现，独行菜种子低温萌发存在低温停滞现象，经短时间较高温度处理能打破停滞。但较高温度处理打破其低温萌发停滞的调控机制不清。本项目拟采用Solexa技术等RNA高通量测序技术及双向电泳技术，分别从mRNA水平和蛋白质水平筛选较高温度处理诱导的差异表达基因，筛选打破独行菜种子低温萌发停滞的相关调控基因；通过实时RT-PCR分析其在较高温度处理前后表达量的变化特性，明确其与打破独行菜低温萌发停滞的相关性；采用转基因技术和RNA干扰技术在拟南芥中超表达或沉默该基因，分析超表达和沉默对种子低温萌发的耐受性影响，阐明独行菜种子低温萌发停滞的分子调控机制，也为揭示其它早春短命植物早春低温萌发的分子机制提供依据。

分布于新疆北部的独行菜(Lepidium apetalum Willd)属典型的早春短命植物，其幼苗期能够在早春的低温条件下生长，具有良好的耐受低温胁迫的能力。前期研究发现，独行菜种子存在萌发停滞现象在低温中，经短时间较高温度处理能打破停滞。但较高温度处理打破其低温萌发停滞的调控机制不清。本项目分别从mRNA水平和蛋白质水平筛选出较高温度处理诱导差异表达显著的基因，筛选打破独行菜种子低温萌发停滞的相关调控基因。通过对转录组分析筛选2108个差异表达基因，其中1118个基因上调表达，990个基因下调表达，通过蛋白质水平筛选出37个差异蛋白点，研究独行菜种子低温萌发停滞解除前后差异表达的蛋白质，分析了3个表达差异显著的蛋白在独行菜种子低温萌发不同时期的表达模式，结果显示差异蛋白基因在转录水平和蛋白水平表达情况基本一致，且这些蛋白参与了表达调控，细胞分裂，氧化还原等多种生理过程。这些基因的表达与解除独行菜种子低温萌发停滞有密切关系，此结果为植物温度信号响应的研究提供了新的线索。通过实时RT-PCR分析LaAP2，LaDREB，laICE1等基因在较高温度处理前后表达量的变化特性，明确其与打破独行菜低温萌发停滞的相关性，发现这些基因在独行菜幼苗期，经低温处理后，其表达量快速显著上调，表明其可能在独行菜幼苗抵抗低温胁迫中起调控作用，为深入探讨独行菜功能基因在独行菜幼苗耐受低温中的作用奠定了基础。采用转基因技术和RNA干扰技术在拟南芥中对LaNHR2B及LaABC1超表达或沉默该基因，表明这些基因的表达对种子低温萌发的耐受性起重要的调控作用。为揭示其它早春短命植物早春低温萌发的分子机制提供依据。