柑橘缺锌响应基因的挖掘及其在缺锌诊断上的应用研究

Zinc deficiency, affecting the yield and quality of citrus fruits, is a common problem in citrus production,. Currently, zinc deficiency could be corrected by foliar application of zinc fertilizer at the early stage of leaf development. However, the leaves at this stage did not frequently show the symptoms of zinc deficiency. The current techniques could not diagnose the zinc deficiency at this stage. As a result, it is difficult to provide the information to spray the zinc fertilizer at the appropriate time only to the zinc dificienct plants. To settle this problem, this study will screen the zinc deficient-responsive genes by genome-wide analysis of gene expression profiling in citrus under zinc deficiency stress based on established hydroponic method. Then the expression patterns of these genes under deficiencies of different nutrient elements will be analyzed to identify the genes that are specially responsive to zinc deficiency. The correlationship between the expression levels of the specific genes and zinc status will be established by detecting the transcript levels of these specific genes at various zinc concentrations, allowing a fast diagnosis of zinc deficiency at the early stage in citrus. This study can not only lay the foundation for the development of a diagnosis technique on zinc deficiency, but also for cloning of zinc deficient-related genes and creation of novel citrus germplasms tolerant to zinc deficiency via genetic engineering in the future, implying that the project is of both theoretic importance and practical potential.

我国柑橘普遍缺锌，影响产量和品质。早期快速准确诊断和及时叶面喷施锌肥是有效矫治柑橘缺锌的重要手段，但现有技术难以达到上述目标，导致防效不佳。为解决此问题，本研究拟在已建立的柑橘缺锌水培体系基础上，对正常和缺锌水培植株叶片样本进行数字化基因表达谱测序，从全基因组范围筛选柑橘缺锌响应基因；再结合这些基因在各种缺素处理下表达模式分析，鉴定特异响应缺锌基因；分析这些特异基因在不同锌营养水平下的表达水平，建立基因表达与锌营养水平间的相关性，以期通过检测缺锌响应特异基因的表达水平来估测柑橘树体锌营养状况，实现柑橘缺锌的早期快速诊断和及时矫治。本项目的开展，既能为开发柑橘缺锌的早期诊断技术提供理论基础，也能为今后克隆缺锌相关功能基因并通过基因工程创制耐缺锌材料奠定基础，既有理论意义也有较大应用价值。

本项目以柑橘主要砧木—枳为材料，研究柑橘缺Zn胁迫下的生理与分子变化，通过数字表达谱测序，挖掘缺Zn特异表达基因，分析基因表达水平与Zn营养水平的相关性，为探索基于基因表达的缺Zn诊断技术奠定基础。主要研究内容及关键结果包括：① 对田间不同缺Zn程度材料的研究表明，缺Zn显著降低净光合速率和叶绿素水平，引起总蛋白含量和抗氧化酶活性变化；② 对枳缺Zn、缺Fe处理4 d、12 d的样品进行了数字表达谱测序，在根、叶中分别筛选到大量差异表达基因（DEGs），包括439个缺Zn叶的DEGs，481个缺Zn根的DEGs，946个缺Fe叶的DEGs和967个缺Fe根的DEGs，其中在4 d和12 d样品中均差异表达的基因可能与缺Zn、缺Fe明显相关；和缺Fe响应的差异表达基因相比，有280个基因特异在缺Zn叶中差异表达； ③ 挑选了15个候选的缺Zn特异表达基因，分析其在对照（CK）、缺Zn（-Zn）、缺Fe（-Fe）、缺锰（-Mn）样品中的表达水平，结果发现4个基因在缺Zn叶片中特异上调表达，鉴定为缺Zn特异表达基因；④ 对枳ZIP家族的12个基因成员进行克隆，分析了其在CK、-Zn、-Fe、-Mn样品中的表达水平，结果发现2个基因在缺Zn叶片中特异上调表达，鉴定为缺Zn特异表达基因；⑤ 研究了上述鉴定的6个缺Zn特异表达基因在不同Zn水平下的表达模式，发现其中4个基因的表达水平与枳叶片Zn营养水平呈现明显相关性。本项目不仅为研究基于基因表达的缺Zn诊断技术提供了参考，而且为解析柑橘缺Zn应答机理奠定了基础。