【作者】 李达；

【导师】 陆建良；

【作者基本信息】 浙江大学 ， 茶学， 2021， 博士

【摘要】 嘌呤生物碱(Purine alkaloids,PAs)是茶树重要的次生代谢产物,主要包括咖啡碱、可可碱、茶碱和苦茶碱等物质,对茶树抗逆性和茶叶品质均有显著作用。茶树咖啡碱合成途径及其相关基因目前已经明晰,而分解代谢途径及关键基因尚不明确。本研究通过对‘福鼎大白茶’、‘凌云白毫’和苦茶(Camellia kucha)不同成熟度叶片进行PAs检测、转录组测序和加权基因共表达网络(Weighted gene co-expression network analysis,WGCNA)分析,获得咖啡碱分解代谢相关的细胞色素P450 82Ds(Cytochrome P450 82Ds,CYP82Ds)等候选基因,采用PCR技术克隆了茶树CYP82Ds基因及其启动子,分析了其表达特性,并利用原核、真核表达系统和反义寡核苷酸技术(Antisense oligonucleotide,as ODN)研究了CYP82D2基因生物学功能,主要结果如下:(1)‘福鼎大白茶’和苦茶叶片的优势生物碱分别为咖啡碱和苦茶碱,其含量均随叶片成熟度增加而下降,而‘凌云白毫’优势生物碱因叶片成熟度而异,即幼嫩叶片中以咖啡碱为主、成熟叶片中以苦茶碱为主、且在叶片成熟过程中两者呈明显的消长关系。从3种茶树不同成熟度样品中筛选出了6839条差异表达基因(Differentially expressed genes,DEGs),表现出19种特异性表达模块,其中pink模块和lightgreen模块与咖啡碱和苦茶碱含量表型显著相关,pink模块中DEGs主要与“卟啉与叶绿素代谢”、“次生代谢物质的合成”和“嘌呤代谢”通路有关,lightgreen模块DEGs则富集在“m RNA监管途径”、“丙酮酸代谢”和“光合生物的碳固定”等通路;pink和lightgreen模块中的关键基因(Hub genes)分别包括环氧化物水解酶B(Epoxide hydrolase B,EPHB)、类金属硫蛋白1(Metallothionein-like protein 1,MT1)、丝氨酸/苏氨酸蛋白激酶(Serine/threonineprotein kinase 3,RKF3)等以及CYP82D、磷酸甘油醛脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase,G3PB)、磷脂酶D(Phospholipase D zeta 2,PLDZ2)。(2)克隆了‘福鼎大白茶’、‘凌云白毫’和苦茶的CYP82D1、CYP82D2、CYP82D3基因,其开放阅读框分别为1572bp、1560bp、1599bp,编码523、519和532个氨基酸残基;CYP82D1和CYP82D2有2个跨膜螺旋,CYP82D3有1个跨膜螺旋;不同茶树资源中CYP82D1和CYP82D3氨基酸序列非常相似,而‘福鼎大白茶’CYP82D2与‘凌云白毫’和苦茶存在50个氨基酸差异。荧光定量PCR结果显示,随着叶片成熟度增加,‘福鼎大白茶’和‘凌云白毫’CYP82D1表达量上调、苦茶CYP82D1表达量下调;CYP82D3在‘凌云白毫’不同成熟度叶片均有表达、且在老叶中表达量最高,在苦茶叶片中无明显表达,在‘福鼎大白茶’不同成熟度叶片之间表达无显著差异;‘凌云白毫’和苦茶中CYP82D2表达量显著高于‘福鼎大白茶’,其中‘凌云白毫’老叶和苦茶成熟叶表达量最高,分别是‘福鼎大白茶’嫩叶的45760倍和101446倍;CYP82D2表达量与苦茶碱含量变化趋势较为一致。启动子克隆和元件分析显示,3种茶树资源CYP82D2基因启动子区域除具有高等植物启动子必备的基础核心作用元件外,存在大量与光响应、环境胁迫响应、激素响应相关的作用元件以及MYB等转录因子结合位点,其中,在‘凌云白毫’和苦茶CYP82D2启动子中类MYB转录因子结合位点数量多于‘福鼎大白茶’,且低温响应元件LTR只存在于‘凌云白毫’CYP82D2启动子区域;不同茶树资源叶片CYP82D2的差异表达可能与启动子的元件差异有关。(3)构建了含‘凌云白毫’CYP82D2和细胞色素P450还原酶(Cytochromes P450 reductase,CPR)基因的酵母共表达载体p AO815-αCYP-αCPR,转入Pichia pastoris GS115菌株,经1%甲醇诱导表达,检测到两个目的蛋白,但浓度很低,不足以支撑酶活分析。构建了p Cold-TF-CYP和p Cold-TF-CPR大肠杆菌表达载体,转入E.coli Origami B(DE3)菌株,经IPTG诱导,两个目的蛋白可在大肠杆菌中正确表达,其中,CYP82D2最佳诱导条件为培养温度15℃、0.4m M IPTG处理时间24h,CPR最佳诱导条件为温度15℃、0.6m M IPTG处理时间24h;在含p Cold-TF-CYP和p Cold-TF-CPR质粒的混合菌液中添加嘌呤生物碱,未检测到底物的变化,后续将进一步调整体外反应体系以验证酶活。此外,亚细胞定位结果表明,CYP82D2定位于内质网。(4)利用了as ODN技术敲低了‘凌云白毫’叶片中CYP82D2的表达,结果发现CYP82D2-as ODN处理的叶片中咖啡碱和可可碱的含量更高,表明CYP82D2参与了‘凌云白毫’叶片中咖啡碱向苦茶碱分解代谢过程。更多还原

【Abstract】 Purine alkaloids(PAs)are important secondary metabolites of tea plant,mainly including caffeine,theobromine,theophylline and theacrine,which have significant effects on stress resistances of tea plant and quality of tea products.The pathway of caffeine biosynthesis and its related genes have been clarified in tea plant,but the catabolic pathway and key genes are still unclear.In this study,cytochrome P450 82Ds(CYP82Ds),the candidate genes related to caffeine catabolism were obtained from leaves with different maturity harvested from ’Fuding Dabaicha’,’Lingyun Baihao’ and Camellia kucha through PAs detection,transcriptome sequencing and weighted gene co-expression network analysis(WGCNA).The main results are as follows:(1)Caffeine and theacrine were the dominant alkaloids in leaves of ’Fuding Dabaicha’and C.Kucha,respectively,and their contents decreased with an increase of leaf maturity.The dominant alkaloids of ’Lingyun Baihao’ varied with leaf maturity,i.e.,caffeine was dominant in young leaves while theacrine did in mature leaves,and an obvious alterantion occurred during leaf maturity.6839 differentially expressed genes(DEGs)were screened from the various maturity leaves harvested from three tea plant resuorces,and 19 modules with different expression patterns were obtained through WGCNA.Among them,pink and lightgreen modules were significantly related to content of caffeine and theacrine,respectively.DEGs in pink module were mainly related to ’porphyrin and chlorophyllin metabolism’,’synthesis of secondary metabolites’ and ’purine metabolism’ pathways;DEGs in lightgreen module were enriched in ’m RNA regulatory pathway’,’pyruvate metabolism’ and ’carbon fixation of photosynthetic organisms’.The candidate hub genes in pink and lightgreen modules included epoxide hydrolase B(EPHB),metallothionein-like protein 1(MT1)and serine/threonine-protein kinase 3(RKF3),as well as CYP82 D,glyceraldehyde-3-phosphate dehydrogenase(G3PB)and phospholipase D zeta 2(PLDZ2),respectively.(2)CYP82D1,CYP82D2 and CYP82D3 genes were cloned from leaves of ’Fuding Dabaicha’,’Lingyun Baihao’ and C.kucha.Their open reading frames were 1572 bp,1560bp and 1599 bp,respectively,encoding 523,519 and 532 amino acid residues.Two transmembrane helices were predicted in CYP82D1 and CYP82D2,while only one in CYP82D3.Amino acid sequences of CYP82D1/3 were similar in different tea plant resources,but 50 amino acids of CYP82D2 in ’Fuding Dabaicha’ were found different in comparion with ’Lingyun Baihao’ and C.kucha.q PCR results showed that the expression of CYP82D1 in ’Fuding Dabaicha’ and ’Lingyun Baihao’ was up-regulated,while that in C.kucha was down-regulated.Expression of CYP82D3 was detected in all sampled leaves of ’Lingyun Baihao’ and the significantly highest level in old leaves,but not witnessed in leaves of C.kucha.CYP82D3 expressed similarly in all leaves of’Fuding Dabaicha’.The expression levels of CYP82D2 in ’Lingyun Baihao’ and C.kucha were significantly higher than those in ’Fuding Dabaicha’,and highest levels were detected in old leaves of ’Lingyun Baihao’ and mature leaves of C.kucha,which were45760 and 101446 times higher than those in tender leaves of ’Fuding Dabaicha’,respectively.The expression trend of CYP82D2 was consistent with that of theacrine content.The element analysis of promoter showed that there were a large number of elements related to light response,environmental stress response,hormone response,MYB and other transcription factor binding sites in the promoter region of CYP82D2 in three tea plant resources,in addition to the essential basic core elements of higher plant promoters.The number of MYB-like transcription factor binding sites in CYP82D2 promoters of ’Lingyun Baihao’ and C.kucha was more than that in ’Fuding Dabaicha’,and low temperature responsive element LTR only existed in ’Lingyun Baihao’.The differential expression of CYP82D2 in leaves of various tea plant resources might be related to the difference of promoter elements.(3)The CYP82D2(CYP)and a cytochromes P450 reductase(CPR)obtained from’Lingyun Baihao’ were inserted into the yeast co-expression vector p AO815 and was designated as p AO815-αCYP-αCPR.The obtained vector was transferred into Pichia pastoris GS115 strain.After being induced by 1% methanol,the two target proteins were achieved,but their concentrations were too low to support the enzyme activity analysis.The expression vectors of p Cold-TF-CYP and p Cold-TF-CPR were constructed and transformed into E.coli Origami B(DE3),respectively.After IPTG induction,the two target proteins could be expressed properl in E.coli.The CYP82D2 was optimally induced with 0.4mm IPTG at 15°C for 24 h,and the CPR was optimally induced with 0.6mm IPTG at 15°C for 24 h.Purine alkaloids were added into the mixed bacterial solution containing vector p Cold-TF-CYP and p Cold-TF-CPR,but no product was detected.In order to verify the enzyme activity of the CYP82D2,the in vitro reaction system should be further adjusted.In addition,subcellular localization test showed that CYP82D2 was located in endoplasmic reticulum.(4)To study the effect of CYP82D2 on caffeine catabolic pathway,antisense oligonucleotide(as ODN)technology was used to knock down the expression of CYP82D2 in tea leaves of ’Lingyun Baihao’.It was found that the content of caffeine and theobromine treated with CYP82D2-as ODN was higher when the expression of CYP82D2 was inhibited,indicating that CYP82D2 participated in the caffeine catabolism process in ’Lingyun Baihao’.更多还原