砀山酥梨果实苯丙氨酸解氨酶基因cDNA克隆及序列分析

为了研究梨石细胞形成与木质素合成的关系,以砀山酥梨果实为材料,通过基于同源性的RT—PCR方法,克隆木质素合成途径中的关键酶苯丙氨酸解氨酶（PAL）基因。同时利用基因数据库资料对克隆的PAL序列进行比较分析。结果表明,从砀山酥梨果实中克隆的PAL基因eDNA片段长度为585bp,推断其编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90％以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。     

额尔齐斯河白斑狗鱼假尾复口吸虫的分子鉴定

利用分子生物学方法对采自额尔齐斯河（中国段）的白斑狗鱼（Esox lucius Linnaeus）晶状体内寄生的复口吸虫（Diplostomum）进行了种类鉴定,结果显示其种类为假尾复口吸虫（D.paracaudum）,为中国新记录种。通过PCR扩增待鉴定种的部分18S-ITS1-5.8S序列,测序后与GenBank中假尾复口吸虫的序列进行比对,发现其相似度达到99.6%。将该序列与7种复口吸虫的ITS1序列进行比对后构建系统进化树,进化树以单殖吸虫Gyrodactylus parvae为外类群,结果显示:待鉴定种与假尾复口吸虫（D.paracaudum）聚为一支,其支持率达到了50%以上,支持它们为同一虫种。     

三七对血液系统作用的研究进展

三七,为五加科（Ａｒａｌｉａｃａｅ）植物人参三七Ｐａｎａｘｎｏｔｏｇｉｎｓｅｎｇ（Ｂｕｒｋ）Ｆ．Ｈ．Ｃｈｅｎ（Ｐ．Ｐｓｅｕｄｏｇｉｎｓｅｎｇ Ｗａｌｌ;Ｐ．ＳａｎｃｈｉＨｏｏ）的干燥根,别名田七．是我国名贵中草药之一,主要产于广西、云南等地。味甘微苦 ,性温。三七含有多种化学成分 ,其中三七皂甙 (简称ＰＮＧ)为主要有效成分之一 ,其含量８%～１２ %。ＰＮＧ中包括三七皂甙Ａ (Ｒ -Ａ) ,三七皂甙Ｂ (Ｒ -Ｂ) ,三七皂甙Ｃ(Ｒ -Ｃ) ,三七皂甙Ｄ (Ｒ -Ｄ１) (Ｒ -Ｄ２) ,三七皂甙Ｅ (Ｒ -Ｅ) ,三七皂甙Ｆ(Ｒ -Ｆ)。经理化常…     

枸骨IcFPS1基因的克隆、表达及生物信息学分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是三萜皂苷生物合途径的一个关键酶,为研究FPS基因在枸骨中的功能,该研究采用PCR技术将一个FPS基因的cDNA序列从枸骨叶中分离出来,并命名为IcFPS1。结果表明:根据测序结果分析发现扩增获得的IcFPS1基因cDNA长度为1 591 bp,包含一个完整的开放阅读框,大小为1 029 bp。通过序列分析发现枸骨IcFPS1基因编码342个氨基酸,分子量和等电点分别为39.58 kDa和5.18。通过理化性质预测分析发现IcFPS1蛋白不含信号肽,不含有跨膜区域,该IcFPS1蛋白为亲水性蛋白质。通过序列多重比对发现IcFPS1蛋白质与其他植物的FPS蛋白质高度同源,有共同的保守区域和氨基酸序列,其中与西洋参FPS序列的相似性高达89%。通过系统进化树分析发现枸骨FPS蛋白与同属于被子植物的五加科植物FPS蛋白亲缘关系较近,说明FPS基因在进化过程中相对比较保守。根据蛋白调控网络预测分析结果发现该蛋白可能与IPP1、IPP2、GGPS3、GGPS6和ERA1相互作用,参与类异戊二烯的合成代谢过程。通过实时荧光定量PCR分析发现IcFPS1基因在枸骨各个组织部位中均有表达,其中在枸骨根中表达量最高,在茎和雌花中表达量最低。     

基于rbcL序列试论鸢尾属部分物种间的系统发育关系

选用叶绿体rbcL基因对鸢尾属植物的分类和系统发育进行了分析。以雄黄兰为外类群,对21份鸢尾属材料和GenBank下载的共49条序列采用最大简约法构建系统进化树,探讨了鸢尾属的分类系统。针对有争议的物种,聚类结果表明：马蔺与白花马蔺关系较远;扁竹兰和蝴蝶花亲缘关系较扇形鸢尾近;细锐果鸢尾和锐果鸢尾亲缘关系近,大锐果鸢尾较之远;四川鸢尾和薄叶鸢尾聚类近;西伯利亚鸢尾系内物种间聚类复杂,亲缘关系不清楚。     

三七鲨烯合酶基因在三七根、茎、芦头中的转录表达与三萜皂苷合成

三萜皂苷是三七、人参等重要药用植物的主要有效成分．采用改进的异硫氰酸胍法提取到三七RNA,经RT-PCR克隆技术获得三七三萜合成通路关键酶之一鲨烯合酶(squalene synthase, SS) cDNA,长1 270 bp,读码框架编码415个氨基酸．比对分析表明,推衍的三七SS氨基酸序列与人参、积雪草、青蒿、拟南芥和水稻SS的一致性分别为98%、89%、81%、78%和71%．利用实时荧光定量RT-PCR技术对三七根、茎、芦头的SS转录表达研究发现,一年生三七根SS基因转录表达量最高,高于茎和芦头的表达;但总皂苷含量是:芦头>叶>根>茎．可能与SS之后其它三萜合成关键酶的活性差异或/和三萜合成后的定向输送及累积有关．     

珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究

珠子参作为名贵中草药已有上千年应用历史,珠子参皂苷是珠子参的主要活性成分,由达玛烷型和齐墩果烷型三萜皂苷组成。目前,对珠子参皂苷的生物合成了解甚少,有必要开展与皂苷合成相关的基础研究工作。已有报道表明,法尼基焦磷酸合酶（FPS）可能是植物中皂苷合成的关键酶。本研究克隆了珠子参FPS基因（PjFPS）的cDNA序列,并对其进行了生物信息学分析。基于PjFPS序列构建了珠子参过表达载体pCAMBIA2300s-PjFPS,将其转化到珠子参细胞中,成功获得阳性转基因细胞;测定了阳性细胞系中PjFPS基因的相对表达量、FPS酶活、皂苷以及植物甾醇含量的变化。结果表明,与野生型珠子参细胞相比,PjFPS转基因珠子参细胞系中,PjFPS基因的相对表达量、FPS酶活以及皂苷含量均有不同程度的提高。而且,由于皂苷合成代谢流的增加,也促进了相关重要关键酶基因的表达。在效果最好的阳性细胞中,PjFPS基因的相对表达量、FPS酶活、皂苷含量分别为对照的12、4和3倍;同时,转基因细胞系的植物甾醇含量也有所升高。本研究通过对皂苷合成途径关键酶基因的调控实现了对皂苷生物合成的调节,为获得高效、稳定的珠子参皂苷合成调控技术提供理论参考和依据。     

羽叶三七的转录组测序与三萜皂苷生物合成的关键酶基因的识别

羽叶三七是五加科人参属的名贵药材,三萜皂苷为羽叶三七最主要的活性成分。为了探索羽叶三七根茎中皂苷物质生物合成的分子基础,采用Illumina Hi Seq 2000高通量测序获得羽叶三七根茎的转录组数据;使用Trinity和TGICL软件实现Uni Gene的de novo拼接;基于BLAST完成Uni Gene的蛋白功能注释、KOG功能注释、GO分类和KEGG代谢通路分析。最终通过de novo拼接注释得到Uni Gene 62 240个。研究发现,羽叶三七根茎部表达的26个Uni Gene与三萜碳环骨架合成相关;三萜合成通路中的关键酶FPS、SS、SE等,分别有11 114个Uni Gene。该研究发现的三萜皂苷合成相关候选基因对于阐明羽叶三七三萜皂苷合成方式研究提供了理论基础。     

澳洲宝石鲈线粒体DNA COI基因的克隆与序列分析

对澳洲宝石鲈线粒体DNA细胞色素氧化酶Ⅰ亚基(cytochrome oxidase subunit Ⅰ,COI)基因进行了扩增、克隆和测序,并对COI序列进行了分析.结果显示,澳洲宝石鲈COI基因序列长度为631 bp,其中A、T、G和C 4种碱基的含量分别为27.7%,23.6%,29.8%和18.9%.与从GenBank中查到的其他4种蜊科鱼类的同源序列比对,邻接法(neighbor-joining)构建系统进化树.结果显示,5种蜊科鱼类聚在一起,分为两个大的支系,其中花身蜊与条纹蜊首先聚成一支,然后与詹氏弱棘鯻和银锯眶鯻聚成的一支共同组成一个支系;而高体革鯻单独聚为一个支系,所得的聚类结果与传统的分类结果基本一致.     

植物二氢黄酮醇4-还原酶基因的研究进展

二氢黄酮醇4-还原酶（DFR）是花青素合成途径的关键酶,在花色的修饰中起重要作用。我们简述了DFR基因及其调节基因的研究进展,构建的系统进化树体现了部分单子叶与双子叶植物间的亲缘关系与进化差异,阐述了DFR调控基因的调控机制及DFR基因的应用前景。     

三七法呢基焦磷酸合酶原核表达载体的构建及其表达

在克隆得到三七法呢基焦磷酸合酶(FPS)cDNA的基础上,构建原核表达载体pET32a(+)/FPS,并转化大肠杆菌BL21(DE3)受体菌,在37℃,1 mmol/L IPTG浓度条件下,诱导表达FPS融合蛋白,对诱导表达条件进行了优化.结果表明:成功构建了原核表达载体pET32a(+)/FPS,并在大肠杆菌中成功表达FPS蛋白,分子量约为40 kD,为进一步开展FPS的蛋白纯化和功能分析奠定了基础.     

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.     

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.     

Roles of BOR1, DUR3, and FPS1 in boron transport and tolerance in Saccharomyces cerevisiae

The roles of three membrane proteins, BOR1, DUR3, and FPS1, in boron (B) transport in yeast were examined. The boron concentration in yeast cells lacking BOR1 was elevated upon exposure to 90 mM boric acid, whereas cells lacking DUR3 or FPS1 showed lower boron concentrations. Compared with control cells, cells overexpressing BOR1 or FPS1 had a lower boron concentration, and cells overexpressing DUR3 had a higher boron concentration. These results suggest that, in addition to the efflux boron transporter BOR1, DUR3 and FPS1 play important roles in regulating the cellular boron concentration. Analysis of the yeast transformants for tolerance to a high boric acid concentration revealed an apparent negative correlation between the protoplasmic boron concentration and the degree of tolerance to a high external boron concentration. Thus, BOR1, DUR3, and FPS1 appear to be involved in tolerance to boric acid and the maintenance of the protoplasmic boron concentration.     

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p,Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD⁺/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.     

Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.     

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua.