基于高密度分子遗传图谱的茶树多酚氧化酶活性QTL定位及其遗传分析

Polyphenol oxidase (PPO) is the key enzyme in the formation of black tea quality during its processing. Screening tea cultivar resources with high PPO activity is an important direction of black tea quality breeding. The purpose of this study is to elucidate the genetic mechanism of tea PPO activity variation and to explore genes or molecular markers closely linked to the key sites of PPO activity. The results will provide a basis for marker-assisted selection breeding of tea and screening of suitable tea cultivars for producing black tea. Marked density of tea genetic map is not saturated because of a limited number of mapping population and molecular markers. So it is hard to identify and dissect quantitative trait loci (QTL) mapping. In this study, an F1 mapping population was derived from an inter-varietal cross between C.sinensis cv.Taoyuandaye (female) and C.sinensis cv.Baihaozao (male). The expression of PPO in gene, protein and enzyme levels in parental tea and F1 population were studied using qRT-PCR, Western blot and UV. Parental genetic maps and the consensus map were constructed separately based on pseudo-testcross theory, using simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphis (MFLP), start codon targeted (SCoT) markers and specific length amplified fragment sequencing (SLAF-seq). Genetic analysis and QTL mapping were conducted for PPO activity in tea with the aim of initiating marker-assisted selection and breeding in tea improvement program.

多酚氧化酶(PPO)是红茶加工中品质形成的关键酶，选育具有高PPO活性的茶树品种资源是红茶品质育种的重要方向。阐明PPO活性变异的遗传机制、挖掘与控制PPO活性关键位点紧密连锁的基因或分子标记，对指导茶树分子育种和选育红茶优良品种具有重要指导意义。由于作图个体和分子标记数量的限制，已构建的茶树遗传图谱标记密度不够饱和，难以开展数量性状位点(QTL)定位等后续研究。本研究拟以项目组前期获得的'桃源大叶'×'白毫早'大规模F1子代群体为材料，采用qRT-PCR、Western blot和UV研究PPO在茶树亲本及F1群体间基因、蛋白和酶活表达情况。采用SSR、AFLP、MFLP、SCoT标记技术和简化基因组测序技术，结合“双假测交”策略分别构建双亲遗传图谱以及双亲整合图谱，并对茶树PPO活性进行遗传分析和QTL定位。项目旨在阐明茶树PPO活性的遗传控制基础，为开展茶树分子育种奠定基础。

多酚氧化酶(PPO)是红茶加工中品质形成的关键酶，选育具有高PPO活性的茶树品种资源是红茶品质育种的重要方向。本项目通过研究调控茶树PPO基因表达的分子机制、挖掘与PPO活性相关的分子标记，对指导茶树分子育种和选育红茶优良品种具有重要指导意义。.本研究应用PCR技术克隆CsPPO1和CsPPO3碱基序列。重组表达菌株在IPTG浓度为0.2mM，诱导温度为18℃，诱导时间为6H，诱导表达效果最佳。茶树CsPPO1和CsPPO2基因分别编码595个氨基酸和612个氨基酸，均含有PPO家族特有的PPO1_DWL结构域、Tyrosinase结构域和PPO1_KFDV结构域。序列分析表明均为亲水性蛋白，不存在信号肽，属于非分泌蛋白。从二级结构分析，两个蛋白都含有α-螺旋、β-折叠和大量的无规则卷曲结构。CsPPO1和CsPPO2基因的启动子分别包含两个和三个E-box元件，即存在可能与bHLH转录因子进行特异性结合的位点。CsMYB12和CsMYB48属于亲水性蛋白，含有1个MYB的功能结构域。CsMYB12、CsMYB48均能在SD/-Trp平板正常生长，CsMYB12和CsMYB48可以正常生长且X-α-Gal活性检测变蓝，结果表明CsMYB12和CsMYB48在酵母体内有转录激活活性。CsbHLH51转录因子全长837bp，编码279个氨基酸，分子量为30.94kDa。构建CsbHLH51-pBD载体，通过REN/LUC比值证明CsbHLH51存在自激活活性，是一个激活子。双荧光素酶报告基因系统实验证明CsbHLH51可能通过与CsPPO1和CsPPO2的启动子上的元件结合对下游基因产生了激活作用。亚细胞定位实验表明CsMYB12、CsMYB48和CsbHLH51定位于细胞核，属于核蛋白。PPO基因中外显子共有1 800个碱基，外显子存在92个SNPs位点，平均每20个碱基出现1个SNP位点，表明PPO基因在所选茶树品种之间的种群间遗传多样性较高。转换类型有A↔G，C↔T，颠换类型有A↔C，A↔T，G↔C，其中62个SNPs属于同义突变，30个SNPs位点为非同义突变，导致氨基酸发生变异，占31.5%。利用不同茶树品种间的PPO基因SNP位点，可以成功鉴别区分开94个品种。高PPO酶活品种桃源大叶和海南大叶在第955位、678位和1522位发生突变。