细胞质转化酶基因CsINV2和CsINV12调控茶树抗寒响应的作用机制

Low-temperature stress seriously restricts the shoot growth of tea plant and the yield in tea production. Cytoplasmic invertase (CIN) can irreversibly hydrolyze sucrose into glucose and fructose, playing a great role in balancing ROS contents. However, the function and mechanism of CsINVs in cold resistance in tea plant are mostly unknown. Our previous study showed that, two cytoplasmic invertase genes, CsINV2 and CsINV12, played a great role in responding to low-temperature stress in tea plant, and the activity of CIN was dramatically induced by low-temperature stress in tea plant. The characteristics of sequence variations and different subcellular localizations demonstrate that CsINV2 and CsINV12 possess different foundation and regulation mechanisms as they involved in cold response of tea plant. In view of this, we propose to detect the expression profiles and the enzyme activity of CsINV2 and CsINV12 in different cultivars with different cold resistance under low temperature stress condition, and then illuminate the relationship between both of them and low temperature; assay the activity and the enzyme’s active sites of their encoded proteins by the technology of functional complementation of yeast mutants; verify their physiological function under low temperature stress using overexpressed transgenic Arabidopsis; combined with promoter responsiveness analysis, investigate the mechanisms of CsINV2 and CsINV12 involved in cold response by using metabolomics and qRT-PCR techniques. This research proposal aims to elucidate the acting mechanism of CsINV2 and CsINV12 in the cold response of tea plant, and provide theoretical basis for breeding tea cultivars with cold resistance and for consummating the study on the cold resistance mechanism of the tea plant.

低温胁迫严重制约着茶树新梢生长和茶叶产量提高。细胞质转化酶能不可逆水解蔗糖生成葡萄糖和果糖，在维持ROS平衡方面具有重要作用，但它们在茶树抗寒响应中的功能和作用机制尚未明确。我们前期研究发现，CsINV2和CsINV12是茶树抗寒响应中两个重要的细胞质转化酶基因，低温处理茶树后二者的酶活性显著升高；序列变异及不同亚细胞定位特性表明，二者参与茶树抗寒响应存在特异的功能和调控模式。本项目拟通过分析低温条件下不同抗寒茶树品种中CsINV2和CsINV12基因的表达和蛋白含量，明确二者与低温胁迫的关系；采用酵母互补技术分析二者编码蛋白的活性及催化位点；利用过表达转基因拟南芥验证二者在低温胁迫下的生理功能；结合启动子分析，采用代谢组和qRT-PCR技术解析二者参与抗寒响应的作用机理。揭示CsINV2和CsINV12基因调控茶树抗寒响应的作用机制，为培育抗寒茶树品种和完善茶树抗寒机理研究提供理论依据。

低温胁迫是茶树生长发育、茶叶品质和产量的重要限制因子，开展茶树抗寒分子机理研究以培育抗寒茶树品种能有效解决这一问题。本项目从表达、功能和表达调控三方面研究了CsINV2和CsINV12基因参与茶树抗寒响应的作用机制。组织特异性分析发现，CsINV2和CsINV12在9种不同茶树组织中均有表达，且在嫩果中具有较高的转录丰度。时空表达模式分析发现，CsINV2和CsINV12在不同处理时间点受低温、干旱和盐胁迫诱导差异表达，特别是受低温诱导持续上调表达。冷驯化条件下，CsINV2和CsINV12在不同抗寒茶树品种中差异表达，表明二者可作为潜在的抗寒茶树品种早期鉴定指标。原核表达分析发现，CsINV2和CsINV12蛋白大小均为70 kD左右，且二者均能将蔗糖水解成葡萄糖和果糖，表明二者均具有转化酶活性。启动子分析发现，CsINV2pro::GUS和CsINV12pro::GUS转基因拟南芥根、茎、叶和花均具有GUS活性，且二者在花中的GUS活性最高；低温处理下，CsINV2pro::GUS和CsINV12pro::GUS转基因拟南芥GUS活性均高于常温处理，表明CsINV2和CsINV12介导了茶树整个生长发育过程，且在茶树抗寒响应中具有重要作用。过表达分析发现，CsINV2和CsINV12均能促进转基因拟南芥早花，而CsINV2和CsINV12过表达转基因烟草植株均小于野生型烟草，表明CsINV2和CsINV12过表达介导了转基因植株中的糖代谢过程。同时，本项目还鉴定到多个参与茶树抗寒响应的CsPMEIs、CsARGs和CsG6PDHs基因，明确了这些基因的组织表达特性和时空表达模式，并验证了CsPMEI2和CsPMEI4在转基因拟南芥中的生理功能。本项目的完成进一步丰富了茶树抗寒分子机制研究，为茶树抗寒分子育种奠定了坚实的理论基础。