Characterizafion of gene network in isoquinoline alkaloid biosynthesis and its application

1. 生物合成酶基因的分离与鉴定(1)黄连小檗碱生物合成中去甲氯嘌呤合成酶和四氢小檗碱氧化酶的鉴定与鉴定(2)黄连花木兰碱生物合成中铜嘌呤合成酶（CYP80G2）的鉴定与鉴定(3)加利福尼亚罂粟血根碱生物合成中stylopine合成酶（CYP719A2/A3）的鉴定与鉴定2。利用分离的生物合成基因合成异喹啉生物碱的代谢工程(1)过表达限速酶基因提高异喹啉生物碱的产量；例如，从日本黄连中分离的去甲氯嘌呤6- o -甲基转移酶在加利福尼亚罂粟细胞中的过表达(2)利用RNAi技术进行基因沉默，通过代谢阻断积累中间体；例如，小檗碱桥酶RNAi载体转化的转基因加利福尼亚罂粟细胞中的网状积累(3)Pr…通过引入新的分支途径产生更多新的代谢物；例如，在加利福尼亚罂粟细胞中引入从黄连中分离的苦参碱9-O-甲基转移酶。利用LC-MS、LC-NMR对转基因细胞的代谢物谱进行了表征。(4)过表达生物合成基因（3′-羟基n -甲基氯嘌呤4′o -甲基转移酶）增加了转基因黄连中异喹啉类生物碱的积累。异喹啉生物碱生物合成转录调控系统的研究(1)黄连原生质体转录因子基因瞬时RNAi筛选系统的建立。(2) WRKY1和bHLH1作为黄连细胞异喹啉生物碱合成的综合性基因调控因子的鉴定(3)黄连异喹啉生物碱合成中部分生物合成基因启动子序列的分离及启动子活性的鉴定。确定了在黄连原生质体中表达的最小启动子序列。(1)利用分离的生物合成基因，在大肠杆菌中重建了从多巴胺到网状碱的异喹啉生物碱生物合成途径。(2)利用带CYP80G2基因及以上基因的芽殖酵母转大肠杆菌，以多巴胺为原料制备木兰花碱

1. Isolation and characterization of biosynthetic enzyme genes(1) Identification and characterization of norcoclaurine synthase and tetrahydroberberine oxidase in berberine biosynthesis from Coptis japonica(2) Identification and characterization of corytuberine synthase (CYP80G2) from Coptis japonica in magnoflorine biosynthesis(3) Identification and characterization of stylopine synthase (CYP719A2/A3) from California poppy in sanguinarine biosynthesis2. Metabolic engineering of isoquinoline alkaloid biosynthesis using biosynthetic genes isolated(1) Increase of the isoquinoline alkaloid production by the overexpression of rate-limiting enzyme gene; e.g., overexpression of norcoclaurine 6-O-methyltransferase isolated from Coptis japonica in California poppy cells(2) Accumulation of intermediate by the metabolic block using gene silencing with RNAi technology; e.g., reticuline accumulation in transgenic California poppy cells transformed with RNAi vector for berberine bridge enzyme(3) Pr … More oduction of novel metabolites by the introduction of new branch pathway ; e.g., introduction of scoulerine 9-O- methyltransferase isolated from Coptis japonica in California poppy cells. Modification of metabolite profiles in transgenic cells has been characterized with LC-MS, LC-NMR.(4) Increase of isoquinolie alkaloid accumulation by the overexpression of biosynthetic gene (3'-hydroxy N-methylcoclaurine 4' O-methyltransferase) was determined in transgenic Coptis japonica plants.3. Characterization of transcriptional regulation system in isoquinoline alkaloid biosynthesis(1) Development of novel screening system to isolate transcriptional factor genes using transient RNAi in Coptis japonica protoplasts.(2) Identification of WRKY1 and bHLH1 as the comprehensive gene regulator specific for isoquinoline alkaloid biosynthesis in Coptis japonica cells(3) Isolation of promoter sequences for some biosynthetic genes in isoquinoline alkaloid biosynthesis in Coptis japonica and characterization of promoter activities. Minimum promoter sequences for the expression in Coptis japonica protoplasts were identified.4. Reconstruction of isoquinoline alkaloid biosynthesis in microbe(1) Using isolated biosynthetic genes, isoquinoline alkaloid biosynthetic pathway from dopamine to reticuline was reconstructed in Escherichia coli.(2) Using budding yeast with CYP80G2 gene and above transgenic E. coli, magnoflorine was produced from dopamine Less