滇龙胆龙胆苦苷生物合成三个基因克

发布时间：2018-03-12 10:38

本文选题：滇龙胆　切入点：3-羟基-3-甲基戊二酰辅酶　出处：《云南中医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的克隆获得滇龙胆龙胆苦苷生物合成途径中可能的三个关键酶基因:3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、香叶醇-10-羟化酶(G10H)和环烯醚萜合酶(IS)的c DNA序列,对三个基因进行原核表达,并分析它们的时空表达量与龙胆苦苷积累的关系。方法从滇龙胆转录组数据中获得HMGR、G10H和IS的转录本,采用RT-PCR方法获得三个基因的全长c DNA序列;利用p EASY-E1/BL21表达系统,对三个基因进行原核表达;运用生物信息学方法对序列进行生物信息学分析;采用实时荧光定量法测定三个基因在滇龙胆的根、茎、叶中分别于苗期、花期、果期和枯萎期的相对表达量;利用超高效液相色谱法测定对应滇龙胆的不同部位于不同生长时期的龙胆苦苷含量;采用SPSS 20软件分析三个基因的相对表达量与对应龙胆苦苷含量的相关性。结果克隆得到两条HMGR基因序列,分别命名为GRHMGR-1和GRHMGR-2。GRHMGR-1全长1747bp,ORF为1605bp,编码534个氨基酸,GRHMGR-2全长1731bp,ORF为1572bp,编码523个氨基酸,两条序列编码的蛋白都有着HMGR典型的多肽位点。一条G10H的c DNA序列,全长1400bp,ORF为1248bp,编码415个氨基酸,命名为GRG10H,保守区域分析显示GRG10H属于P450家族;一条长1746bp的基因组G10H序列,内含子预测显示其含三个外显子和两个内含子。一条全长1388bp的IS基因,ORF为1176bp,共编码391个氨基酸,定名为GRIS,保守区域分析显示GRIS属于NADB Rossmann超家族成员。GRHMGR-1、GRHMGR-2、GRG10H和GRIS成功在大肠杆菌中分别表达出58KDa、56 KDa、48 KDa和44 KDa的目的蛋白。GRHMGR和GRG10H在滇龙胆不同生长时期的根、茎和叶中都有表达;GRIS只在滇龙胆不同生长时期的茎和叶中被检测到表达,根中并没有检测到表达。测定结果显示滇龙胆根中的龙胆苦苷含量随着年限的增长而逐年增长,而茎和叶中的则随着生长时期的变化逐渐减少。结论克隆得到滇龙胆可能的关键酶基因HMGR、G10H和IS并构建了原核表达系统,为后期真核表达系统的建立提供了一定的依据。GRHMGR、GRG10H和GRIS的表达量与龙胆苦苷含量积累不具有明显相关性。
[Abstract]:Objective to clone the c DNA sequences of three key enzyme genes in the biosynthesis pathway of gentiopicrin from Gentianopicrin: HMGRG, geraniol-10-hydroxylase (G10H) and iridoid synthase (ISS). Three genes were expressed in prokaryotes and the relationship between their spatiotemporal expression and the accumulation of gentiopicrin was analyzed. Methods the transcripts of HMGRG G10H and is were obtained from the transcriptional data of Dian Gentiana, and the full-length c DNA sequences of the three genes were obtained by RT-PCR method. Using p EASY-E1/BL21 expression system, three genes were expressed in prokaryotes, bioinformatics methods were used to analyze the sequence, and real-time fluorescence quantitative method was used to detect the three genes in root, stem and leaf of Gentiana chinensis at seedling stage. The relative expression of gentiopicrin in flowering, fruit and withering stages was determined by ultrahigh performance liquid chromatography (HPLC), and the contents of gentiopicrin in different parts of Dian gentian at different growth stages were determined by ultrahigh performance liquid chromatography. SPSS 20 software was used to analyze the correlation between the relative expression of three genes and the corresponding gentiopicrin content. Results two HMGR genes were cloned and sequenced. GRHMGR-1 and GRHMGR-2.GRHMGR-1 were named as 1 747 BP ORF and 1605 BP, respectively, encoding 534 amino acids GRHMGR-2, encoding 1 572 BP, encoding 523 amino acids. The two sequences encoded proteins with typical polypeptide sites of HMGR. A G10H c DNA sequence with a total length of 1400bp1 ORF was 1248 BP, encoding 415 amino acids. Named GRG10H, the conserved region analysis showed that GRG10H belonged to the P450 family, a 1746bp genome G10H sequence, which was predicted to contain three exons and two introns, and a 1388bp is gene with an ORF of 1176bpencoding 391 amino acids. The conserved region analysis showed that GRIS belongs to the NADB Rossmann superfamily. GRHMGR-2 GRG10H and GRIS successfully expressed the target proteins. GRHMGR and GRG10H of 58K Daji56KDahu48 KDa and 44 KDa in different growth stages of Gentiana yunnanensis, respectively. The expression of GRIS was only detected in stems and leaves of Gentiana chinensis at different growth stages, but not in roots. The results showed that the content of gentiopicrin in the roots of Gentiana davidiana increased year by year with the increase of years. Conclusion the key enzyme genes HMGRG G10H and is were cloned and the prokaryotic expression system was constructed. There was no significant correlation between the expression of GRHMGRGRG10H and GRIS and the accumulation of gentiopicrin.
【学位授予单位】：云南中医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.239

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