摘要
【目的】毛竹叶中含有大量具有很强生物活性的黄酮碳苷,C-糖基转移酶(CGT)是黄酮碳苷生物合成代谢途径中C-糖基化的关键酶。本研究从毛竹叶中分离纯化CGT,研究毛竹叶黄酮碳苷的C-糖基化途径以及CGT的酶学性质和一级结构氨基酸序列特征,为后续深入研究毛竹叶中的CGT奠定良好的基础。【方法】通过硫酸铵分层沉淀、透析、葡聚糖凝胶过滤、阴离子交换层析、超滤脱盐等方法纯化CGT,使用SDS-PAGE进行检测。分别用圣草素查尔酮、圣草素、木犀草素作为底物,根据已建立的CGT催化的C-糖基化反应体系,进行可能的C-糖基化途径验证。运用Q-TOF检测分析和数据库搜索比对等方法,确认毛竹CGT的基因序列和一级结构氨基酸序列。【结果】毛竹叶黄酮碳苷CGT的分子量大约是50 kDa,酶反应体系最佳的反应时间是40 min,最佳反应温度是28℃,缓冲盐的最佳pH8.1,底物木犀草素的最佳浓度是31.76μmol·L^(-1)。底物为木犀草素的反应体系在CGT催化下大量转化生成了异荭草苷,底物为圣草素查尔酮和圣草素的反应体系在CGT催化下大量转化生成了未知产物,只有少量转化生成异荭草苷。根据蛋白质质谱裂解规律和蛋白质碎片离子,解析出4个CGT肽段,并均可与毛竹基因(PH01000603G0510)编码的蛋白质氨基酸序列匹配,经数据搜索匹配,毛竹基因(PH01000603G0510)与水稻CGT基因(FM179712)匹配率为81%。【结论】通过对毛竹叶黄酮碳苷C-糖基转移酶提取、分离纯化及质谱鉴定得到可能的毛竹叶黄酮碳苷CGT基因序列(PH01000603G0510)。确定毛竹叶黄酮碳苷C-糖基化途径,主要途径是CGT催化木犀草素和UDP-葡萄糖直接合成异荭草苷,次要途径是CGT催化圣草素查尔酮和UDP-葡萄糖,或圣草素和UDP-葡萄糖间接合成异荭草苷。毛竹叶CGT极易催化生成C-6黄酮苷(异荭草苷),而极少催化生成C-8黄酮苷(荭草苷)。
【Objective】C-glycosyl flavonoids are abundant in Phyllostachys edulis leaves and possess vital biological activities.C-glycosyltransferase(CGT)is the key C-glycosylation enzyme in C-glycosyl flavonoid biosynthesis.However,no reports currently exist on the C-glycosylation pathway and C-glycosyl flavonoid CGT from P.edulis leaves.In this work,The main aims of this work were to analyse and confirm the predominant C-glycosylation pathway,enzymatic properties,primary amino acid sequence and gene sequence of CGT and to provide a good foundation for further studying CGT from P.eduli s leaves.【Method】The CGT of a C-glycosyl flavonoid from P.edulis leaves was first purified by ammonium sulfate fractionation precipitation,dialysis,gel chromatography and diethylaminoethyl anion exchange,and detected using SDS-PAGE.Three possible substrates(eriodictyol chalcone,eriodictyol and luteolin)were selected as substrates to analyse the possible C-glycosylation pathways using the established C-glycosylation reaction system and to confirm which pathway was predominant.Using Q-TOF analysis and database searching,the gene and amino acid sequences of P.edulis CGT were analysed and confirmed.【Result】The results suggested that the molecular weight of P.edulis CGT is about50kDa,and the optimal incubation time,incubation temperature,buffer pH and substrate(luteolin)concentration for the CGT reaction system was40min,28℃,8.1and31.76μmol·L^-1,respectively.A large amount of the luteolin underwent CGT-catalysed C-glycosylation was converted into isoorientin in reaction system3,while a large amount of eriodictyol chalcone and eriodictyol in reaction system1and2were converted into unknown products,only a small portion was converted into isoorientin.According to fragmenting law and the diagnostic fragments of the protein peptide ions,four CGT peptide ions were analysed and confirmed,which was aligned with the amino acid sequence encoded by the gene from P.edulis gene(PH01000603G0510),the amino acid sequence of the four CGT peptide ions perfectly matched the amino acid sequence encoded by the P.edulis gene(PH01000603G0510).The matching rate of P.edulis gene sequence(PH01000603G0510)with rice(Oryza sativa Japonica Group)CGT gene sequence(PH01000603G0510)was81%.【Conclusion】Through separation,purification and structural identification of CGT from P.edulis,the possible gene(PH01000603G0510)and amino acid sequences of C-glycosyl flavonoid CGT from P.edulis leaves were analysed and confirmed.The C-glycosylation pathways of flavonoids in P.edulis were also determined.The main C-glycosylation pathway of isoorientin in P.edulis leaves is via the direct synthesis of isoorientin from luteolin and UDP-glucose,and the secondary pathway is via the indirect synthesis of isoorientin from eriodictyol chalcone and UDP-glucose,or eriodictyol and UDP-glucose.The C-glycosyl flavonoid CGT in P.edulis leaves catalyzes the accumulation of flavone6-C-glucosides(isoorientin)but seldom catalyzes the accumulation of flavone8-C-glucosides(orientin).
作者
王源杰
郭雪峰
赵蕾
郭成
王煜炜
Wang Yuanjie;Guo Xuefeng;Zhao Lei;Guo Cheng;Wang Yuwei(International Centre for Bamboo and Rattan,Beijing 100102)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2018年第12期60-69,共10页
Scientia Silvae Sinicae
基金
国家自然科学基金项目"毛竹黄酮碳苷合成代谢途径中C-糖基化及其C-糖基转移酶的研究"(31570345)
关键词
毛竹
C-糖基转移酶
C-糖基化
异荭草苷
木犀草素
Phyllostachys edulis
C-glycosyltransferase
C-glycosylation
isoorientin
luteolin
