两种植物二萜合酶基因的研究
其他题名Research on Diterpene Synthase Genes from Two Plants
李军玲
学位类型硕士
导师曾英
2009-05-18
学位授予单位中国科学院昆明植物研究所
学位授予地点昆明植物研究所
学位专业植物学
关键词毛萼香茶菜 古巴焦磷酸 贝壳杉烯氧化酶 米仔兰 Cdna文库 二萜合酶 后修饰
摘要论文第一章为毛萼香茶菜对映-贝壳杉烯类二萜化合物生物合成途径中两个关键酶基因CPS(copalyl pyrophosphate synthase)和KO(kaurene oxidase)的克隆。毛萼香茶菜(Isodon eriocalyx (Dunn) Kudo)属唇形科(Labiatae)香茶菜属(Isodon)植物,其主要的化学成分是对映-贝壳杉烯类二萜(ent-kaurenoids)化合物,具有抗菌、消炎、抗肿瘤活性,但其生物合成途径尚不清楚。我们推测CPS催化GGPP环化生成CDP(Copalyl pyrophosphate)作为合成ent-kaurene的前体,KO则催化ent-kaurene氧化为多种二萜。根据已报道的CPS和KO氨基酸保守序列,分别设计简并引物,用RT-PCR方法获得了基因特异片段,包括2条CPS类似基因(IeCPS1和IeCPS2,核苷酸相似度为69%)特异片段,经blastx在线序列比对,IeCPS1基因片段与甜叶菊(Stevia rebaudiana)的CPS相似度为70%,IeCPS2基因片段与野甘草(Scoparia dulcis)CPS相似度为78%;一条KO类似基因(IeKO)特异片段,与甜叶菊(Stevia rebaudiana)、草莓(Fragaria grandiflora)KO相似度分别为70%、71%;然后设计基因特异引物做RACE-PCR,IeCPS1获得了全长cDNA序列;IeKO和IeCPS2获得了完整的3’ 端,相应的5’ RACE实验仍在进行中。 论文第二章是米仔兰二萜合酶基因的克隆。米仔兰(Aglaia odorata Lour)属楝科(Meliaceae)米仔兰属(Aglaia),实验所选的研究材料为温室下经过多代繁殖的栽培苗,其形态结构和其次生代谢产物均已发生很大的变化,其中海兔烷型(dolabellane)二萜化合物是栽培苗才产生的次生代谢产物。本文对海兔烷(dolabellane)的生物合成途径进行了探讨,并根据已经发表的萜类合酶基因,设计简并引物,通过RT-PCR方法获得了1条萜类合酶类似基因特异片段,与其它植物萜类合酶相似度在47%左右。利用SMART技术构建了叶片cDNA文库并尝试RACE-PCR,但均没有获得目的基因全长。 论文第三章是综述部分,详述了萜类生物合成途径相关萜类合酶近期的研究进展,重点综述了萜类后修饰酶,包括氧化酶、双键还原酶、酰基转移酶、糖基转移酶。
其他摘要In Chapter 1, we demonstrate the isolation of two key enzyme genes, named CPS (copalyl pyrophosphate synthase) and KO (kaurene oxidase) respectively, in the ent-kaurenoid biosynthesis pathway from Isodon eriocalyx (Dunn) Kudo. The main products isolated from Isodon eriocalyx are ent-kaurenoids, which possess antibacterial, antiinflammation and anticancer activities. Up to now, ent-kaurenoid biosynthesis pathway is still unclear. Here we speculate on the pathway. GGPP is cyclized by CPS to form CDP (copalyl pyrophosphate) as the precursor of ent-kaurene, while KO is responsible for the oxidation of ent-kaurene. An RT-PCR method using degenerate primers based on the consensus sequences of plant CPSs and KOs allowed the isolation of two kinds of CPS-like cDNA fragments (IeCPS1 and IeCPS2,69% identities at nucleotide level ) and one KO-like cDNA fragment (IeKO ). By blastx on line, we find that IeCPS1 fragment shows 70% identities with Scoparia dulcis while IeCPS2 fragment shows 78% identities with Stevia rebaudiana, and that IeKO fragment shows 70% identities with Stevia rebaudiana and 71% identities with Fragaria grandiflora. Then we designed gene specific primers to perform RACE-PCR. We have got full-length cDNA sequence of IeCPS1; complete 3’ end sequences of IeCPS2 and IeKO have also been obtained , yet experiments about 5’-end sequences of them are still under way. Chapter 2 is about the cloning a special diterpene synthase gene from Aglaia odorata Lour. We choose Aglaia odorata Lour cultured in green house after couples of generations as our experimental material, which has great changes in its morphology and secondary metabolites compared with wild type, such as dolabellane diterpenes only existing in cultispecies. The biosynthesis of the unusual diterpenes is discussed. We performed RT-PCR using degenerate primers based on the consensus sequences of some plant terpene synthases, and obtained one terpene synthase-like gene cDNA fragment showing about 47% identities with other plant terpene synthase. We also constructed a leaf cDNA library using SMART method and performed RACE-PCR, but did not got the full sequence of target gene. The last part reviews the advances regarding the terpene synthases involved in the biosynthesis of terpene in plants, wih emphasis on the post-modification enzymes, including oxidases, double-bond reductases, acylases and glycosyltransferases.
页数107
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/422
专题昆明植物所硕博研究生毕业学位论文
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李军玲. 两种植物二萜合酶基因的研究[D]. 昆明植物研究所. 中国科学院昆明植物研究所,2009.
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