代谢工程改造酿酒酵母生产β-胡萝卜素

β-胡萝卜素在食品、药品和化妆品领域有广泛用途。为获得生产β-胡萝卜素的微生物细胞工厂,本研究首先在酿酒酵母BY4742中过表达甲羟戊酸(MVA)途径的限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因及二萜化合物合成的关键酶牻牛儿基牻牛儿基焦磷酸合酶(GGPS)基因,来提高牻牛儿基牻牛儿基焦磷酸(GGPP)的供给。在酿酒酵母底盘菌BY4742-T2的基础上整合来源于成团泛菌和红法夫酵母的β-胡萝卜素合成基因,比较酿酒酵母工程菌生产β-胡萝卜素的差别。结果表明提高酿酒酵母中HMGR和GGPS酶基因的表达能将工程菌中β-胡萝卜素的产量提高26.0倍。另外,来源于真核生物红法夫酵母的合成基因相比成团泛菌,更有利于酿酒酵母生产β-胡萝卜素。最终获得的酿酒酵母工程菌BW02能生产1.56 mg/g细胞干重的β-胡萝卜素,为进一步获得高产β-胡萝卜素细胞工厂提供基础。     

西瓜GGPS基因果实特异性表达载体构建与遗传转化

[目的]将牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase,GGPS)基因转化西瓜。[方法]利用RT-PCR技术克隆西瓜果实GGPS基因CDS区;构建由果实特异性启动子AGPL1调控GGPS基因、以nptⅡ为筛选标记基因的植物表达载体,并转入农杆菌EHA105;通过农杆菌介导法对西瓜品种"红一号"进行遗传转化。[结果]经过卡那霉素筛选和PCR初步鉴定,获得4株转基因阳性植株。[结论]构建了GGPS基因果实特异性表达载体,并将其转入西瓜品种"红一号"中,转化效率为0.13%。     

不同花期‘西伯利亚’百合花瓣单萜合成途径转录组分析（英文）

以‘西伯利亚’百合(Lilium‘Siberia’)花蕾期、半开期、盛开期、衰败期的花瓣为材料,利用RNA-seq技术对其转录组进行高通量测序,分析单萜合成途径中差异表达的基因并阐明其分子机制。结果显示,‘西伯利亚’百合通过转录组测序分析共得到56.28 Gb clean base,223.40 Mb clean reads和124 233个unigene,其中35 749个基因得到注释。萜骨架合成途径中的基因表达水平在不同花期表现出显著差异。其中,甲基赤藓糖醇磷酸(MEP)中的1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)、1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR)、4-羟基-3-甲丁-2-烯基二磷酸合成酶(HDS)、4-羟基-3-甲丁-2烯基二磷酸还原酶(HDR)、牻牛儿基二磷酸合成酶(GPS)基因的表达水平随花期变化呈先升高后降低的趋势。罗勒烯合成酶(OCS)基因表现出相似变化规律,在盛开期表达量最高。甲羟戊酸(MVA)途径中的3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)的基因表达同样出现先升高后降低的趋势。单萜合成下游的分支途径中,茄尼基二磷酸合成酶(SDS)、牻牛儿基牻牛儿基二磷酸合成酶(GGDR)基因的表达则出现相反的趋势,在盛开期的表达量最低。研究结果表明MEP途径中的关键基因可随花期变化规律性的表达,以调控单萜的生物合成,在盛开期有较高释放量,且盛开期MVA途径的活化以及泛醌和萜醌代谢支路基因的低表达也促进了单萜的生物合成。     

西瓜牻牛儿基牻牛儿基焦磷酸合成酶基因的克隆及表达分析

目的:了解牻牛儿基牻牛儿基焦磷酸合成酶基因(GGPS)在4种不同瓤色西瓜果实发育过程中的表达变化,将为西瓜类胡萝卜素基因操作提供工具。方法:通过RT-PCR和RACE技术从西瓜果实中克隆GGPS的cDNA全长,用生物信息学方法分析其序列及推测氨基酸序列,并用实时定量PCR技术检测GGPS在不同瓤色果实发育过程的表达水平。结果:GGPS基因cDNA全长1 445 bp(GenBank登录号为KF914758),其开放阅读框为1 092 bp,编码363个氨基酸。预测其氨基酸序列N末端存在转运肽信号序列。西瓜GGPS与甜瓜和黄瓜GGPS的序列同源性高达90%以上。GGPS在红瓤西瓜中的表达量最高,白瓤西瓜中最低。结论:克隆获得的GGPS可能对西瓜果实中类胡萝卜素的积累具有重要影响。     

盐胁迫条件下玉米萜类合成相关基因的表达分析

萜类是植物生长发育中具有重要作用的次级代谢产物,某些萜类可被生物和非生物胁迫诱导产生。用200 mmol/L NaCl溶液处理商用玉米品种天塔五号（F1）及其父母本三叶期幼苗,qRT-PCR结果发现萜类合成途径中的萜烯合酶基因2（terpene synthase 2, TPS2）、萜烯合酶基因3（terpene synthase 3, TPS3）、牻牛儿基牻牛儿基焦磷酸合成酶基因4（geranylgeranyl diphosphate synthase 4, GGPS4）在三组材料中表达量随胁迫时间延长均先上调再下调,且F1表达量显著高于亲本。F1自交F2代耐盐性及基因表达情况分析表明,F2代中三个基因表达仍与耐盐性呈正相关。对盐胁迫条件下总类胡萝卜素及各成分含量、生物量、光合指标、叶绿素和脯氨酸含量进行测定,发现F1耐盐能力明显优于亲本,表明 TPS2、TPS3、GGPS4 基因高水平表达与F1发挥耐盐优势、抵御逆境胁迫有一定相关性。     

不同花期‘西伯利亚’百合花瓣单萜合成途径转录组分析

以‘西伯利亚’百合（Lilium ‘Siberia’）花蕾期、半开期、盛开期、衰败期的花瓣为材料,利用RNA-seq技术对其转录组进行高通量测序,分析单萜合成途径中差异表达的基因并阐明其分子机制。结果显示,‘西伯利亚’百合通过转录组测序分析共得到56.28 Gb clean base,223.40 Mb clean reads和124 233个unigene,其中35 749个基因得到注释。萜骨架合成途径中的基因表达水平在不同花期表现出显著差异。其中,甲基赤藓糖醇磷酸（MEP）中的1-脱氧-D-木酮糖-5-磷酸合成酶（DXS）、1-脱氧-D-木酮糖-5-磷酸还原异构酶（DXR）、4-羟基-3-甲丁-2-烯基二磷酸合成酶（HDS）、4-羟基-3-甲丁-2烯基二磷酸还原酶（HDR）、牻牛儿基二磷酸合成酶（GPS）基因的表达水平随花期变化呈先升高后降低的趋势。罗勒烯合成酶（OCS）基因表现出相似变化规律,在盛开期表达量最高。甲羟戊酸（MVA）途径中的3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGR）的基因表达同样出现先升高后降低的趋势。单萜合成下游的分支途径中,茄尼基二磷酸合成酶（SDS）、牻牛儿基牻牛儿基二磷酸合成酶（GGDR）基因的表达则出现相反的趋势,在盛开期的表达量最低。研究结果表明MEP途径中的关键基因可随花期变化规律性的表达,以调控单萜的生物合成,在盛开期有较高释放量,且盛开期MVA途径的活化以及泛醌和萜醌代谢支路基因的低表达也促进了单萜的生物合成。     

茉莉酸甲酯对灵芝三萜生物合成的影响及其灵芝应答基因的差异表达

灵芝三萜类化合物是灵芝的主要药用成分之一,灵芝三萜含量的多少是衡量灵芝质量高低的重要指标。经研究发现茉莉酸甲酯能显著提高灵芝三萜含量并上调灵芝三萜生物合成途径中的羟甲基戊二酰辅酶A还原酶、法尼酯焦磷酸合成酶和羊毛甾醇合成酶等基因的表达。通过在灵芝中建立的cDNA-AFLP体系,采用64对引物组合对灵芝的转录组进行分析。64对引物组合共产生3 910个TDFs。茉莉酸甲酯不同诱导条件下约有919个TDFs的表达谱发生了变化,占表达谱的23.5%,其中上调表达的有703个,下调表达的有216个。选择458个TDFs进行回收、TA克隆、测序后,获得390个高质量的TDFs片段。通过蛋白数据库同源性检索,并对蛋白功能进行GO分类,结果表明:390个高质量的TDFs中,BlastX比对后没有显著匹配的序列占77.1%。在有匹配蛋白的序列中,与碳代谢及能量相关的基因为最大一组,占35.2%,转录调控类及信号传导类基因分别占14.3%和11.0%。通过荧光定量PCR技术考察了茉莉酸甲酯对20个基因转录水平的影响,与cDNA-AFLP分析的结果相一致。并通过灵芝基因过量表达技术和基因沉默技术对筛选到的相关基因进行沉默和过量表达,研究筛选到的基因在灵芝三萜生物合成与调控中的作用,获得大量参与灵芝三萜生物合成与调控的候选基因,进一步阐明了茉莉酸甲酯诱导下灵芝基因表达的变化图。     

三角褐指藻dxs基因克隆与诱导表达

为研究6种外源因素处理对三角褐指藻(Phaeodactylum tricornutum)1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)基因表达的影响,通过高通量转录组测序技术获得三角褐指藻dxs基因cDNA全长序列,并对其进行生物信息学分析。研究结果表明,三角褐指藻dxs基因cDNA全长2476 bp, ORF全长2193 bp,编码730个氨基酸,具有高度保守的ThDP结合位点和转酮醇酶结构域。三角褐指藻DXS蛋白为亲水性稳定蛋白,相对分子质量(M_w)为79.31 kD,理论等电点为6.65,具有信号肽、跨膜区域、卷曲螺旋和TM-螺旋等。系统进化树分析结果表明, DXS蛋白进化树分为高等植物和藻类2个分支,高等植物DXS蛋白进一步分为DXS1和DXS2两支,藻类DXS蛋白聚类为3个分支,分别为硅藻门、红藻门和绿藻门分支,三角褐指藻聚类在硅藻门分支上。诱导表达调控结果表明,三角褐指藻dxs基因受到茉莉酸甲酯(MeJA)、花生四烯酸(AA)、硫酸铈铵(ACS)、光合诱导素(PIF)、光合抑制剂(DCMU)和光质等6种外源因素的诱导调控。在100μmol/L MeJA...     

刺五加HMGR基因的表达及其对皂苷合成的影响

以actin为内参基因,采用Real time PCR法分析产地、发育时期、器官以及茉莉酸甲酯(MeJA)对刺五加HMGR基因表达的影响,并采用分光光度法测定皂苷含量,利用SPSS 17.0软件分析两者的相关性。结果表明:HMGR基因在各器官中均有表达,相对表达量间差异较明显(p0.05)。刺五加HMGR基因在盛花期表达量最大,叶片衰老期表达量最低。Me JA处理后显著提升了萌芽期至盛花期HMGR的表达量,而对果实快速生长期之后的影响较小。不同产地刺五加的HMGR基因表达量和总皂苷含量差异显著,而HMGR基因的表达量与总皂苷含量同升同降,存在极显著的正相关关系(p0.01)。     

高等植物牻牛儿基牻牛儿基焦磷酸合成酶基因的研究进展

牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)在植物体内催化牻牛儿基牻牛儿基焦磷酸(GGPP)的合成,GGPP是萜类物质、类胡萝卜素、叶绿素及几个重要植物激素合成的前体物,是联系植物体内多条重要次生代谢通路的节点物质。本文综述了植物GGPPS基因近年来的生物学功能研究进展和该基因家族的遗传分类情况,以及GGPPS小亚基基因的重要调控作用,拟为深入研究植物GGPPS基因的生物学功能和萜类含量调控的遗传工程提供新认识和新思路。     

Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana

Isoprenoid biosynthesis in plants occurs by two independent pathways: the cytosolic mevalonate (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. In this study, we investigated the cellular effects of depletion of IspE, a protein involved in the MEP pathway, using virus-induced gene silencing (VIGS). The IspE gene is preferentially expressed in young tissues, and induced by light and methyl jasmonate. The GFP fusion protein of IspE was targeted to chloroplasts. Reduction of IspE expression by VIGS resulted in a severe leaf yellowing phenotype. At the cellular level, depletion of IspE severely affected chloroplast development, dramatically reducing both the number and size of chloroplasts. Interestingly, mitochondrial development was also impaired, suggesting a possibility that the plastidic MEP pathway contributes to mitochondrial isoprenoid biosynthesis in leaves. A deficiency in IspE activity decreased cellular levels of the metabolites produced by the MEP pathway, such as chlorophylls and carotenoids, and stimulated expression of some of the downstream MEP pathway genes, particularly IspF and IspG. Interestingly, the IspE VIGS lines had significantly increased numbers of cells of reduced size in all leaf layers, compared with TRV control and other VIGS lines for the MEP pathway genes. The increased cell division in the IspE VIGS lines was particularly pronounced in the abaxial epidermal layer, in which the over-proliferated cells bulged out of the plane, making the surface uneven. In addition, trichome numbers dramatically increased and the stomata size varied in the affected tissues. Our results show that IspE deficiency causes novel developmental phenotypes distinct from the phenotypes of other MEP pathway mutants, indicating that IspE may have an additional role in plant development besides its role in isoprenoid biosynthesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Genbank accession number for IspE: ABO87658.     

Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

棉花miR397 LAC4模块调控棉铃虫抗性研究

miRNA(microRNA)通过调控其靶标基因在植物的生长、发育和抗逆过程中扮演着重要的角色。该研究采用分子生物学和生物化学等方法,探讨棉花miR397 LAC4参与植株木质素生物合成和对棉铃虫抗性响应机制。结果发现：(1)棉花miR397(ghr miR397)在转录后调控漆酶基因(GhLAC4)的表达,GhLAC4属于蓝铜氧化酶家族,通过调控木质素合成,抵御棉铃虫入侵棉花。(2)GUS报告基因融合表达和酶活性测定表明,ghr miR397在转录后切割靶标基因GhLAC4抑制其表达。(3)利用VIGS(virus induced gene silencing)技术在棉花中沉默和过表达ghr miR397,棉铃虫抗性检测分析表明,沉默miR397表达会增加棉花对棉铃虫的抗性,但过表达ghr miR397则会降低棉花的抗性。(4)选择性和非选择性棉铃虫实验分析、组织化学染色和木质素含量测定表明,沉默GhLAC4表达会减少木质素的积累,增加棉花对棉铃虫的敏感性。研究表明,ghr miR397 GhLAC4模块共同微调棉花木质素合成来参与棉花抗虫性调控,同时也为棉花抗虫育种提供了新思路。     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by wounding and methyl jasmonate

HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase; E.C.1.1.1.34) supplies mevalonate for the synthesis of many plant primary and secondary metabolites, including the terpenoid component of indole alkaloids. Suspension cultures of Camptotheca acuminata and Catharanthus roseus, two species valued for their anticancer indole alkaloids, were treated with the elicitation signal transducer methyl jasmonate (MeJA). RNA gel blot analysis from MeJA treated cultures showed a transient suppression of HMGR mRNA, followed by an induction in HMGR message. Leaf disks from transgenic tobacco plants containing a chimeric hmgl::GUS construct were also treated with MeJA and showed a dose dependent suppression of wound-inducible GUS activity. The suppression of the wound response by MeJA was limited to the first 4 h post-wounding, after which time MeJA application had no effect. The results are discussed in relation to the differential regulation of HMGR isogenes in higher plants.Abbreviations GUS -glucuronidase - hmg gene of hmgr - HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase - JA jasmonic acid - MeJA methyl jasmonate - MUG methylumbelliferyl--d-glucuronide - TDC tryptophan decarboxylase - SDS sodium dodecyl sulfate - SS strictosidine synthase