Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream produ...Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants.展开更多
目的克隆阳春砂萜类生物合成途径上游关键酶——3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase,HMGR)(EC:1.1.1.34)的编码基因;分析基因的功能及其在阳春砂不同组织中的表达。方法用基于RT-PCR的方...目的克隆阳春砂萜类生物合成途径上游关键酶——3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase,HMGR)(EC:1.1.1.34)的编码基因;分析基因的功能及其在阳春砂不同组织中的表达。方法用基于RT-PCR的方法从阳春砂叶片中获得编码HMGR的cDNA全长序列,克隆基因编码区;用生物信息学的方法对其编码蛋白进行相似性检索和功能分析;用半定量RT-PCR法比较基因在阳春砂不同组织中的表达差异。结果获得了全长2 023 bp的编码阳春砂HMGR的cDNA序列,命名为AvHMGR(GenBank登记号:FJ455511)。AvHMGR编码的蛋白与其他植物来源的HMGR有很高相似性,含有NADPH结合基序和底物HMG-CoA结合基序,N-端有两个跨膜结构域。保守功能结构域的分析结果表明AvHMGR属于3-羟基-3-甲基戊二酰辅酶A还原酶家族。AvHMGR在包括茎、根、果皮和种子团的广泛组织中表达,且在这些组织中的表达量均高于在叶片中的表达。结论从阳春砂中克隆了AvHMGR基因,为进一步鉴定基因功能、探明阳春砂萜类生物合成的基因调控机制打下基础。展开更多
The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number var...The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.展开更多
The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR) is the last step key enzyme of the methylerythritol phosphate(MEP) pathway,synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphat...The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR) is the last step key enzyme of the methylerythritol phosphate(MEP) pathway,synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate,which is important for regulation of isoprenoid biosynthesis.Here the full-length cDNA of HDR,designated TwHDR(GenBank Accession No.KJ933412.1),was isolated from Tripterygium wilfordii for the first time.TwHDR has an open reading frame(ORF) of 1386 bp encoding461 amino acids.TwHDR exhibits high homology with HDRs of other plants,with an N-terminal conserved domain and three conserved cysteine residues.TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant.Since loss-of-function E.coli hdr mutant is lethal,the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant.This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme.The expression of TwHDR was induced by methyl-jasmonate(MJ) in T.wilfordii suspension cells.The expression of TwHDR reached the highest level after 1 h of MJ treatment.These results indicate that we have identified a functional TwHDR enzyme,which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T.wilfordii.展开更多
Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)hav...Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.展开更多
文摘Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants.
基金This work was supported by the National Natural Science foundation of China(81072988).
文摘The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.
基金supported by the National Natural Science Foundation of China(Nos.81422053 and 81373906 to Wei Gao,and No.81325023 to Luqi Huang)the National High Technology Research and Development Program of China(863 Program,No.2015AA0200908)
文摘The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase(HDR) is the last step key enzyme of the methylerythritol phosphate(MEP) pathway,synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate,which is important for regulation of isoprenoid biosynthesis.Here the full-length cDNA of HDR,designated TwHDR(GenBank Accession No.KJ933412.1),was isolated from Tripterygium wilfordii for the first time.TwHDR has an open reading frame(ORF) of 1386 bp encoding461 amino acids.TwHDR exhibits high homology with HDRs of other plants,with an N-terminal conserved domain and three conserved cysteine residues.TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant.Since loss-of-function E.coli hdr mutant is lethal,the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant.This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme.The expression of TwHDR was induced by methyl-jasmonate(MJ) in T.wilfordii suspension cells.The expression of TwHDR reached the highest level after 1 h of MJ treatment.These results indicate that we have identified a functional TwHDR enzyme,which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T.wilfordii.
基金supported by the National Natural Science Foundation of China(No.81260622)Chongqing Natural Science Foundation Project(cstc2018jcyjAX0267).
文摘Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.
