山茶花应答低温胁迫的相关基因发掘及功能研究

Camellia, with evergreen leaves and beautiful flowers,is one of the important evergreen flower trees blossoming in winter and spring.The majority of camellia cultivars can not live through in winter outdoors in the north of the Yangtze River.Low temperature stress is one of the main factors to restrict the camellia industry development.Utilization of cold-resistant resources is the basis of camellia genetic improvement and the important route of ustilizing resistant resouces. In this project, high cold-resistant Camellia japonica 'Qiaofen'will be used as the plant material. Transcriptome sequencing analysis will be carried out using leaves without low temperature treatment.Then, the leaves under low temperature treatment(4℃) will be collected at different treatment time points for the analysis of the expression sequencing profiles.By bioinformatics analysis,some related genes in response to low temperature stress will be obtained and through their expression patterns analysis using Real-time PCR and RNA in situ hybridization,some genes in response to low temperature intensely would be selected as candidate genes.Next,the candidate genes will be transformed to Arabidopsis thaliana and Petunia hybrida.By the tests of cold resistance capacity of transgenic plants, the function of candidate genes will be studied.This project will reveal molecular mechanism of cold-resistant camellia, discovery related gene, and provide cold-resistant gene resources for camellia molecular breeding.

茶花叶绿花娇，是重要的冬春季节开花的常绿观花树种之一，大部分茶花品种在长江以北难以裸地越冬，低温胁迫成为制约其产业发展的主要因素之一。利用耐寒资源发掘基因是茶花遗传改良的基础或是利用抗性资源的重要途径。本项目拟以前期鉴定的山茶花耐寒品种'俏粉'（Camellia japonica'Qiaofen'）为试材，通过对未低温处理叶片的转录组测序和4℃低温处理不同时间的叶片表达谱测序，通过生物信息学分析，发掘山茶花应答低温胁迫的相关基因，利用Real-time PCR验证其表达模式，筛选部分低温应答强烈的基因。利用拟南芥、矮牵牛转化鉴定候选基因的抗寒能力。本项目揭示山茶花耐寒分子机理，挖掘抗寒性相关基因，为山茶花抗寒分子育种提供基因资源。

茶花叶绿花娇，是重要的冬春季节开花的常绿观花树种之一，大部分茶花品种在长江以北难以裸地越冬，低温胁迫成为制约其产业发展的主要因素之一。利用耐寒资源发掘基因是茶花遗传改良的基础或是利用抗性资源的重要途径。本项目以耐寒山茶花品种‘绛雪’为材料，采集低温驯化2 h, 8 h, 24 h, 72 h, 168 h及对照植株叶片进行RNA-seq测序分析，获得了山茶花叶片转录组及低温驯化下的表达谱信息。测序共产生clean reads 1006 M，将所有数据进行Trinity拼接，得到转录本367620个，以此作为后续分析的参考序列。进一步筛选得到低温驯化2 h, 8 h, 24 h, 72 h及168 h差异基因4235、3458、14676、19908和12802个。通过GO注释及KEGG代谢路径分析，发现大量转录因子基因、蛋白激酶基因、激素信号转导途径的基因、不饱和脂肪酸合成及茉莉酸合成相关基因在山茶花低温驯化过程中差异表达。通过对多个具不同耐寒性的山茶品种的进一步分析发现，α-亚麻酸及茉莉酸合成这两个代谢路径可能在山茶低温应答中起重要作用。目前已经成功克隆了11个可能参与α-亚麻酸及茉莉酸合成途径的候选基因并构建植物表达载体；其中CjSAD1和CjSAD2已经获得转化拟南芥T1代阳性苗并进行初步功能验证。本项目揭示了山茶花耐寒分子机理，挖掘得到了抗寒性相关的生物学路径和关键基因，为山茶花抗寒分子育种提供了理论支持和基因资源。