GS1基因家族成员在茶树铵、硝偏性利用中的功能解析

Improper application of nitrogen (N) fertilizer in tea plantation, resulted in a negative effect on tea quality and environmental conditions, is one of the problems which commonly occur in tea cultivation. To solve the problem mentioned above, it is necessary to explore the functional roles of key genes which restrict the process of N assimilation. Glutamine synthetase (GS) is a key enzyme regulating nitrogen assimilation in plants. In our previous study on the transcriptome data mining, 8 GS1 unigenes were obtained. Meanwhile, each GS1 unigene showed distinct expression level under NH4+ and NO3- treatments. This result suggests that genes for GS1 play different roles in the assimilation of NH4+ and NO3-, which may have relationship with the preference assimilation for NH4+ in tea plant. In this project we will isolate the full cDNA length of GS1 unigenes and confirmed the expression patterns of GS1 genes in response to NH4+ and NO3- treatments during the time course and their tissue specificity in tea plant. Subcellular and cellular localization of GS1 genes would suggest their potential functions in the assimilation of NH4+ and NO3-. The role of 2-3 genes will also be investigated in planta by over-expression in callus of tea plant. Expected results from this project will help dissect the functional roles of the GS1 genes in assimilation of NH4+ and NO3-, and consequently provide insight into the mechanisms behind the preference assimilation of nitrogen in tea plants. And also help to improve the N fertilization manner in tea plantation.

茶园氮肥不合理施用是当前茶叶生产亟待解决的重要问题，不仅严重影响茶叶品质形成也对茶园生态带来巨大挑战。而茶树氮素利用效率直接决定了茶园氮肥需求和用量。因此，挖掘茶树氮素利用的关键基因及其功能是提高茶树氮素利用效率和指导茶园合理施肥的首要任务。谷氨酰胺合成酶（GS）是茶树利用氮素的限速酶，申请人前期通过转录组分析获得8个GS1 unigenes，且发现GS1基因家族各成员在茶树氮素利用过程中存在功能差异，可能与茶树铵（铵态氮NH4+）、硝（硝态氮NO3-）偏性利用相关。本项目拟采用基因克隆、基因表达模式分析、细胞学定位、茶树愈伤组织超量表达验证、拟南芥突变体回复试验等手段，明确茶树GS1基因家族成员、表达模式，及各成员在茶树铵、硝偏性利用过程中的生物学功能。为解析茶树氮素偏性利用、提高茶树氮素利用效率提供理论支撑，进而指导茶园氮肥的施用。

茶园氮肥不合理施用是当前茶叶生产亟待解决的重要问题，不仅严重影响茶叶品质形成也对茶园生态带来巨大挑战。而茶树氮素利用效率直接决定了茶园氮肥需求和用量。因此，挖掘茶树氮素利用的关键基因及其功能是提高茶树氮素利用效率和指导茶园合理施肥的首要任务。谷氨酰胺合成酶（GS）是植物利用氮素的限速酶，项目负责人前期通过转录组数据分析获得8个GS1（胞质型谷氨酰胺合成酶）unigenes且在不同氮信号处理下表达存在显著差异，推测其可能参与调控茶树对铵、硝的偏性利用。本项目通过RACE法确定茶树GS1基因家族成员三个CsGS1.1/1.2/1.3；细胞学定位显示其编码蛋白均定位在细胞质。半定量和定量PCR分析CsGS1s组织特异性，结果显示：CsGS1.2在茶树各组织的相对表达丰度显著低于CsGS1.1/1.3，且在叶中表达显著高于茎、根；CsGS1.1在根中的表达显著高于叶，在叶中的表达高于茎；CsGS1.3在根中的表达显著高于茎和叶。不同成员对NH4+/NO3-响应模式分析显示：在根中，CsGS1.1/1.2只受NH4+特异诱导表达，而CsGS1.3在两种氮形态下均被显著抑制；在叶中，CsGS1.2/1.3只受NH4+特异诱导表达，而CsGS1.1表达水平不受NH4+的影响。超表达植物材料NH4+、NO3-利用生物学功能评价证实CsGS1.1和CsGS1.3对铵的同化存在差异，CsGS1.1的过表达可提高植物对铵的耐受力，而CsGS1.3的过表达抑制植物对铵的同化能力。本项目的实施明确了茶树GS1编码基因家族成员及其在茶树不同氮源利用中存在功能差异，为解析茶树氮素偏性利用营养特性和提高茶树氮素利用效率提供理论支撑。已发表第一标注论文5篇，其中SCI 4篇、中文核心期刊1篇，申报发明专利1项。