牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

产紫杉醇内生真菌枝状枝孢霉MD2的发酵条件优化

[目的]通过优化内生真菌枝状枝孢霉MD2的发酵条件,提高10-去乙酰巴卡亭Ⅲ (10-DAB)和紫杉醇(Taxol)的产量.[方法]采用单因素试验分析不同的培养基初始pH值、培养温度、摇床转速和培养时间对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养条件;以YES为基本培养基,采用单因素试验和正交试验分析添加苯甲酸钠、苯丙氨酸、丝氨酸和甘氨酸4种前体物对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养基组分.[结果]优化后发酵条件为:在初始pH为5.0的300 mL YES培养基中,添加15 mg/L苯甲酸钠、25 mg/L苯丙氨酸、5 mg/L丝氨酸、15 mg/L甘氨酸,接种1 mL枝状枝孢霉MD2的孢子悬液(107-10s个孢子/mL),28.0℃、220 r/min发酵培养12d.在此条件下,枝状枝孢霉MD2的生物量、10-DAB和紫杉醇的产量分别为15.5 g/L、471.5 μg/L和569.5 μg/L,与初始发酵条件相比,分别提高了1.3、3.6和3.4倍.[结论]首次获得了枝状枝孢霉MD2生产10-DAB和紫杉醇的较适摇瓶发酵条件,可为进一步放大发酵培养提供参考.     

千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

紫杉醇生物合成相关酶基因的克隆与表达

以牛儿基牛儿基二磷酸为前体的紫杉醇生物合成大约有20步酶促反应,其反应过程已基本阐明,近一半的相关酶基因已得到克隆与表达。综述了编码参与紫杉醇生物合成的紫杉二烯合酶、紫杉二烯5α羟化酶、紫杉烷10β羟化酶、紫杉烷13α羟化酶、紫杉二烯5α醇O乙酰基转移酶、紫杉烷2α苯甲酰转移酶、去乙酰基巴卡亭Ⅲ10βO乙酰转移酶、3氨基3苯基丙酰转移酶和3N去苯甲酰2脱氧紫杉醇苯甲酰转移酶等9个酶基因的克隆和表达方面的研究情况,并指出随着紫杉醇生物合成的分子生物学研究的不断深入,利用分子生物技术大规模生产紫杉醇将为期不远 。     

从南方红豆杉中筛选和鉴定25株产紫杉烷的内生真菌

为从南方红豆杉(Taxus chinensis var.mairei)中分离产紫杉烷的内生真菌,从其幼茎、树皮和叶片中分离纯化了491株内生真菌,经筛选获得25株内生真菌具有产紫杉烷的能力,其中,4株可产紫杉醇、巴卡亭Ⅲ和10-去乙酰巴卡亭Ⅲ,8株能产紫杉醇和巴卡亭Ⅲ,1株能产紫杉醇和10-去乙酰巴卡亭Ⅲ,1株能产巴卡亭Ⅲ和10-去乙酰巴卡亭Ⅲ,6株仅产紫杉醇,5株仅产巴卡亭Ⅲ。根据内生真菌的来源,幼茎中有11株产紫杉烷的内生真菌,叶片中有9株,而树皮中仅有5株。这些菌株的紫杉醇、巴卡亭和10-去乙酰巴卡亭Ⅲ产量分别为0.64~9.87、0.48~3.42和0.20~1.00μg L~(–1)。因此,南方红豆杉中具有紫杉烷类代谢途径的内生真菌来源广,数量多,是研究真菌中紫杉烷类化合物代谢途径的良好材料,也为紫杉烷类抗癌药生产提供了潜在的真菌种源。     

榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因Pa GGPPS(Gen Bank登录号为KM881430)的c DNA开放阅读框(Open reading frame,ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98k D,等电点为6.168,表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。Pa GGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。     

枝孢霉丝孢真菌生长特性的研究

为明确枝孢霉丝孢真菌生物学特性,根据枝孢霉属当前分类系统,选取6株代表菌株,通过单因素分析法,研究了不同培养温度、生长pH和培养基中葡萄糖、蛋白胨浓度对该类菌种生长的影响,并通过微量肉汤稀释法检测了 5种常用表面活性剂对该类菌种的最低抑菌浓度.结果表明,6株枝孢霉真菌适宜生长温度为20～25℃,pH为5.0～6.5,葡...     

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。     

代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。     

枝孢样枝孢霉的研究概况

引起暗色丝孢霉病的枝孢霉属,属半知菌亚门、丝孢菌纲、暗色孢科。该属50余种。枝孢样枝孢霉为腐生真菌,广泛存在于自然界的土壤、某些动物的粪便、蔬菜、腐木、鸟巢、腐烂水果中。人因皮肤外伤接触后有被感染的机会。它也是真菌过敏症的重要病原菌之一。目前对枝孢样枝孢霉的系统报道较少,现就其生理生化代谢、生态学、病理学及临床和抗真菌研究等方面进行综述。     

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.     

Identification of an Abscisic Acid Gene Cluster in the Grey Mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.     

A strategy to identify positional candidate genes conferring Marek''s disease resistance by integrating DNA microarrays and genetic mapping

Marker-assisted selection (MAS) to enhance genetic resistance to Marek's disease (MD), a herpesvirus-induced T cell cancer in chicken, is an attractive alternative to augment control with vaccines. Our earlier studies indicate that there are many quantitative trait loci (QTL) containing one or more genes that confer genetic resistance to MD. Unfortunately, it is difficult to sufficiently resolve these QTL to identify the causative gene and generate tightly linked markers. One possible solution is to identify positional candidate genes by virtue of gene expression differences between MD resistant and susceptible chicken using deoxyribonucleic acid (DNA) microarrays followed by genetic mapping of the differentially-expressed genes. In this preliminary study, we show that DNA microarrays containing approximately 1200 genes or expressed sequence tags (ESTs) are able to reproducibly detect differences in gene expression between the inbred ADOL lines 63 (MD resistant) and 72 (MD susceptible) of uninfected and Marek's disease virus (MDV)-infected peripheral blood lymphocytes. Microarray data were validated by quantitative polymerase chain reaction (PCR) and found to be consistent with previous literature on gene induction or immune response. Integration of the microarrays with genetic mapping data was achieved with a sample of 15 genes. Twelve of these genes had mapped human orthologues. Seven genes were located on the chicken linkage map as predicted by the human-chicken comparative map, while two other genes defined a new conserved syntenic group. More importantly, one of the genes with differential expression is known to confer genetic resistance to MD while another gene is a prime positional candidate for a QTL.