植物类黄酮异戊烯转移酶基因的研究
高娟
学位类型硕士
导师曾英
2010-05
学位授予单位中国科学院研究生院
学位专业植物学
摘要论文第一章为类黄酮异戊烯转移酶基因的克隆和功能鉴定。传统中草药淫羊藿(Epimedium)为小檗科(Berberidaceae)淫羊藿属植物,异戊烯黄酮醇苷化合物含量甚高,其中icariin和epimedinA-C为主要有效成分常被作为药材品质的评价指标。此外淫羊藿还含有微量的breciflavone B。我们推测淫羊藿异戊烯类黄酮的生物合成中,类黄酮异戊烯转移酶催化异戊烯基取代类黄酮(naringenin 、kaempferol或apigenin)A环8位或3'位以及5'位碳上的质子。本实验首先用LC/MS测定栽培于昆明植物园粗毛淫羊藿(Epimedium acuminatum Franch)一年内不同生长时期叶片中 icariin的含量,发现3月份变色时期的幼叶icariin含量最高,提取3月份幼叶的总RNA合成1st cDNA为模板,运用相似性克隆原理,克隆到2条类似异戊烯转移酶基因,一条全长基因EaPT1,ORF1224bp,另一条基因Ts4只得到3'端序列,约940bp。使用大肠杆菌异源表达系统,将EaPT1的全长和去N端信号肽序列分别构建到表达载体pET32a(+)中,在表达菌Rosetta-gamiB(DE3)、RosettaTM 2(DE3)plysS、BL21(DE3)plysE、BL21(DE3)gold和BL21(DE3)中都没有表达出目标融合蛋白。换用酿酒酵母表达系统,pYES2作为表达载体,也没有表达出目标蛋白。用naringenin为底物,LC/MS没有检测到预计底物。论文第二章为毛萼香茶菜(Isodon eriocalyx (Dunn) Kudo)两条CPS(copalyl pyrophosphate synthase)基因IeCPS7、IeCPS11的异源表达与功能鉴定。利用异源表达技术,将此两条基因的全长和去N端信号肽的序列构建到pET32a(+)中,在BL21(DE3)中表达。CPS7去N端信号肽的序列表达出了可溶性融合蛋白,104699.41D,等电点为5.87,融合蛋白共编码924个氨基酸。以GGPP为底物,去焦磷酸后GC/MS检测到了GGPP的环化产物。体外酶反应最适温度为37℃,最适pH为7.1,Mg2+促进反应进程,Mn2+抑制反应进程。CPS11在表达菌Rosetta-gamiB(DE3)、Rosetta 2(DE3)plysS、BL21(DE3)中都只能表达出包涵体。论文第三章为综述部分,综述了近年来植物和真菌中芳香族异戊烯转移酶的分子生物学研究进展,主要包括参与质体醌合成的homogentisate solanesyltransferase、参与维生素E合成的homogentisate phytyltransferase、类黄酮异戊烯转移酶(flavonoid prenyltransferase)以及可溶性的真菌吲哚异戊烯转移酶等。
资助项目In Chapter 1, we isolated a flavonoid prenyltransferase-like gene from traditional Chinese medicinal herb, Epimedium L. (berberidaceae). Epimedium species have a high content of the prenylated flavonol glycosides. Icariin and epimedin A, B and C are frequently used as marker compounds for the quality control of Epimedium. Here we speculate prenyl flavonoids biosynthesis pathway in Epimedium: The flavonoid prenyltransferase is responsible for the prenylation of flavonoids (naringenin 、kaempferol or apigenin) at the 8-position or 3' or 5'-position. Leaves of Epimedium acuminatum Franch in the nursery were collected every month, and then detected the icariin content. The results show that leaves in March have the highest icariin content. Total RNA was extracted from leaves in March as template. A similarity-based cloning strategy yielded a flavonoid prenyltransferase-like gene, named EaPT1. In E. coli. expression system, pET32a(+) was chosen as the expression vector for use in Rosetta-gamiB(DE3)、RosettaTM 2(DE3)plysS、BL21(DE3)plysE、BL21(DE3)gold and BL21(DE3) cells. The full length ORF and truncated sequence were ligated with pET32a(+). We did not detect the target protein in SDS-PAGE. In Saccharomyces cerevisiae expression system, the full length ORF was ligated with pYES2. In this expression system, we still could not detect the protein in SDS-PAGE. LC/MS did not detect the activity of prenyltransferase, with naringenin as substrate. Chapter 2 describes functional expression and characterization of two copalyl pyrophosphate synthase gene from Isodon ericalyx (Dunn) Kudo, named IeCPS7 and IeCPS11. Their full length ORF and truncated sequence were ligated into pET32a(+). These vectors were used to transform E.coli BL21(DE)3. The truncated IeCPS7 sequence expressed a soluble His-tag recombinant protein, 104699.41D, pI5.87, 924aa. The recombinant protein was characterized for diterpene synthase activity by using geranylgeranyl diphosphate(GGPP) as substrates and subsequent GC/MS analysis of products. The purified recombinant IeCPS showed optimum activity at pH7.1. In addition, IeCPS showed maximum activity at 30℃. The enzymatic activity was increased by addition of MgCl2 to the reaction mixture. Unexpectedly, MnCl2 actually inhibited the enzyme activity. In addition,only insoluble recombinant proteins were expressed for IeCPS11 in BL21(DE)3, Rosetta-gamiB(DE3) and RosettaTM 2(DE3)plysS. The last part reviews the advances in molecular studies of aromatic prenyltransferase in plants and fungi, focusing on membrane-bound homogentisate prenyltransferses, flavonoid prenyltransferases as well as soluble indole prenyltransferases.
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/16186
专题昆明植物所硕博研究生毕业学位论文
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高娟. 植物类黄酮异戊烯转移酶基因的研究[D]. 中国科学院研究生院,2010.
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