交替氧化酶基因（ClAOX）SNP位点调控西瓜非生物胁迫响应的内在机制

Abiotic stresses, like cold, is major limited factors that affect plant growth and development, of which its regulating mechanism is focused by researchers. In higher plant, alternative oxidase (AOX) consists of AOX1 and AOX2 subfamily. We have previously identified only one allele for AOX through degenerated PCR amplification from cultivated, wild and relative species and genome sequence Blast analysis in watermelon. Phylogenetic tree indicated that ClAOX is belonged to AOX2 subfamily. Bioinformatics analysis showed a SNP associated with abiotic stress within ClAOX among different subspecies. In this project, we try to study the regulatory mechanism on ClAOX as AOX1 subfamily function in response to abiotic stresses and as AOX2 subfamily function in developmental stage. We firstly construct ClAOX-GFP and ClAOX-GUS to study its expression patterns in relation to abiotic stresses and development. We then construct ClAOX-Knockout and ClAOX-Overexpression lines to proof its biological function associated with abiotic stresses and development. Afterwards, we plan to study regulatory mechanism through protein expression, oxidation-reduction status and post-translation under abiotic stresses. We attempt to reveal the regulatory mechanism on how the single nucleotide polymorphism of ClAOX to regulate abiotic stress in watermelon.

非生物胁迫（如低温等）是影响西瓜生长发育的重要因子，植物交替氧化酶基因（AOX）由AOX1和AOX2亚家族组成，其调控的非生物胁迫机制一直是该领域的研究热点。课题组前期发现西瓜基因组仅编码一个AOX基因，且属于AOX2亚家族，不同亚种之间存在一个与非生物胁迫功能相关的SNP位点。本研究在此基础上，运用遗传学、分子生物学和生物化学等手段，研究ClAOX是否同时具有AOX1和AOX2亚家族相关功能。首先，通过构建ClAOX基因GFP、GUS等，研究ClAOX响应非生物胁迫及生长发育相关功能；然后构建西瓜ClAOX基因的基因敲除（ClAOX-KO）和过量表达（ClAOX-OX）系，验证ClAOX具有调控非生物胁迫与生长发育功能；然后，从蛋白水平上研究在非生物胁迫条件下，ClAOX基因的蛋白表达、氧化还原态和翻译后调控机制，最终阐明西瓜ClAOX基因的SNP位点调控非生物胁迫响应的机制。

交替氧化酶(AOX)是高等植物、真菌、藻类和一些原核生物抗氰呼吸途径的重要成分。高等植物一般包含AOX1和AOX2两个亚家族。本项目通过系统研究首次发现了在西瓜基因组中只存在一个ClAOX基因，该基因同时具有AOX2亚家族的组成性表达特征和AOX1亚家族的低温诱导性；在不同西瓜亚种的ClAOX序列上发现的一个单核苷酸多态性(SNP)，导致了一对ClAOX等位基因；利用拟南芥中异位表达研究验证了该对等位基因导致了植物低温抗性差异；基于不同西瓜亚种间AOX基因差异开发可育种利用的耐寒分子标记。本项目成果为葫芦科作物的抗逆分子设计育种提供了基因资源和理论基础。