Isolation,sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.Abbreviations ALA 5-aminolevulinic acid - copro coproporphyrin - coprogen coproporphyrinogen - protogen IX protoporphyrinogen IX - UROD uroporphyrinogen decarboxylase - uro uroporphyrin - urogen uroporphyrinogen     

The effects of Rhodiola rosea extract on 5-HT level, cell proliferation and quantity of neurons at cerebral hippocampus of depressive rats

The purpose of this study was to investigate the effects of Rhodiola rosea extract and depression on the serotonin (5-HT) level, cell proliferation and quantity of neurons at cerebral hippocampus of depressive rats induced by Chronic Mild Stress (CMS). Seventy male Sprague-Dawley rats were divided into seven groups (10 per group): normal control group, untreated depressive rat model group, negative control group, positive control group, low dosage Rhodiola rosea extract (1.5 g/kg) group, medium dosage Rhodiola rosea extract (3 g/kg) group and high dosage Rhodiola rosea extract (6 g/kg) group. After the depressive rats induced by CMS had received Rhodiola rosea extract for 3 weeks, the 5-HT levels at cerebral hippocampus were detected by high performance liquid chromatography. Bromodeoxyuridine (BrdU) was injected in vivo to label the proliferating cells at hippocampus, and morphometry was used to count the hippocampal neurons. The results showed that the 5-HT level of the three experimental groups had recovered to normal status. The immunohistochemistry of hippocampus BrdU positive cells had returned to the normal level in the group of depressive rats with low dosage Rhodiola rosea extract. In conclusion the results demonstrated that Rhodiola rosea extract could improve 5-HT level in hippocampus in depressive rats, and low dosage Rhodiola rosea could induce neural stem cell proliferation at hippocampus to return to normal level, repairing the injured neurons at hippocampus.     

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Summary Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.     

Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea. Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation (F_ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.     

一种由粉红粘帚霉引起的青稞根腐病

[背景]根腐病在青稞生产中的危害日趋严重,阻碍了青稞根腐病的有效防控及青海省青稞产业的发展。然而人们对青稞根腐病的研究甚少且病原菌不详。[目的]明确青稞根腐病发生的危害、病原及致病性,为青稞根腐病的防控提供理论依据。[方法]采用常规的组织分离法分离青稞根腐病病原,通过形态鉴定与分子鉴定结合的方法对病原进行鉴定,并采用烧杯水琼脂法测定其致病性。[结果]共分离得到4株青稞根腐病病原菌,鉴定为Clonostachys rosea,有较强的致病性且致病性差异显著,经柯赫氏法则验证为青稞根腐病病原菌,并且是一种新的青稞根腐病病原,该类根腐病也是一种新的根腐类病害,在国内外属首次发现。[结论]Clonostachys rosea可引起青稞根腐病且致病性强。     

Biological control by Clonostachys rosea as a key component in the integrated management of strawberry gray mold

Gray mold, caused by Botrytis cinerea, is an important strawberry disease. As gray mold control is difficult, there is a need to evaluate integrated methods to successfully manage the disease. The efficiency of integrating Clonostachys rosea sprays, fungicide sprays, and crop debris removal to manage gray mold was evaluated in field experiments conducted in 2006 and 2007. Leaf colonization by C. rosea (LAC), average number of B. cinerea conidiophores (ANC), gray mold incidence in both flowers (Iflower) and fruits (Ifruit), and yield were evaluated weekly. In both years, LAC was higher in the treatments with no fungicide. When compared to the check, ANC, Iflower and Ifruit were most reduced in treatments that included C. rosea sprays. Maximal reductions were achieved with the combination of C. rosea sprays, fungicide sprays and debris removal (96.62%, 86.54% and 65.33% reductions of ANC, Iflower and Ifruit, respectively). Otherwise, maximal yield (103.14% increase as compared to the check) was achieved with the combination of the three treatments. With just C. rosea sprays, ANC, Iflower and Ifruit were reduced by 92.01%, 68.48% and 65.33%, respectively, whereas yield was increased by 75.15%. Considering the individual effects, application of C. rosea was the most efficient treatment. Chemical control was effective only in plots without debris removal. Elimination of crop debris was the least effective method in reducing gray mold incidence in both flowers and fruits. The integrated control approach enhanced the efficacy of the individual methods of gray mold control and provided high strawberry yield. An important component of this integrated approach it the biological control with C. rosea.     

狭叶红景天幼苗对水分及遮阴的生长及生理生化响应

研究植物对水分和遮阴胁迫的响应及其生理机制对制定合理的栽培管理措施十分必要。以红景天属植物为研究对象,设置土壤含水量分别为田间持水量的80%(过湿水分)、70%(正常水分)、60%(轻度干旱)、40%(中度干旱)、20%(重度干旱)5个水分梯度;设置2个遮阴处理,以全光照(遮阴率为0)为对照、黑色遮阴网遮阴(遮阴率为85%),研究狭叶红景天生长及生理生化指标的变化特征。结果表明:在不同水分处理下,与对照相比,叶绿素含量、茎干重和茎重比(SMR)显著增加(P0.05),株高、总生物量、叶面积、叶干重、叶重比(LMR)、比叶面积(SLA)、叶面积比(LAR)和叶面积根干重比(LARMR)增加,根冠比和根重比(RMR)减少;随着干旱程度加剧,丙二醛(MDA)、脯氨酸(Pro)和可溶性糖(Ss)含量增加,超氧化物歧化酶(SOD)活性总体呈先增加后减小的趋势。在遮阴处理下,株高、SMR、SLA、LAR和LARMR显著增加(P0.05),叶绿素SPAD值和叶面积增加,总生物量、根干重、根冠比和LMR显著减少(P0.05),茎干重和叶干重减少,MDA含量显著增加,Pro含量略有下降,Ss含量减少。在水分胁迫下,狭叶红景天中度干旱时通过增加酶活性抵御伤害,重度干旱超过其阈值,SOD活性下降,植物体受到伤害,Ss可能是主要的渗透调节物质。在遮阴处理下,狭叶红景天通过增加SLA避免遮阴伤害。狭叶红景天在受到环境胁迫时会通过形态改变、调节MDA含量、抗氧化酶活性和渗透调节物质来保证自身正常的生长发育。     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.     

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.     

蛹虫草磷酸甲羟戊酸激酶和焦磷酸甲羟戊酸脱羧酶基因的功能分析

【目的】确定蛹虫草甲羟戊酸途径中的2个关键酶——磷酸甲羟戊酸激酶(CmErg8)和焦磷酸甲羟戊酸脱羧酶(CmErg19)的功能及其对麦角甾醇和虫草素含量的影响。【方法】通过生物信息学分析鉴定蛹虫草中CmErg8和CmErg19,并采用酵母互补确定其功能是否保守;以蛹虫草尿嘧啶营养缺陷型CmΔpyrG为背景菌株,利用农杆菌介导的转化方法对CmErg8和CmErg19进行过表达,观察其对麦角甾醇和虫草素含量的影响。【结果】CmErg8和CmErg19不能互补酵母erg8和erg19突变体的温度敏感表型;CmErg8和CmErg19过表达菌株中麦角甾醇和虫草素含量均有所增加,特别是CmErg19基因过表达可以使虫草素含量提升5倍左右。【结论】本研究揭示了蛹虫草CmErg8和CmErg19的功能,并且发现蛹虫草麦角甾醇合成通路基因可能会影响虫草素含量。     

西藏大花红景天RcCATs与RcSODs基因克隆及功能分析

为探究过氧化氢酶(CAT)和超氧化物歧化酶(SOD)基因家族成员在西藏大花红景天高原恶劣生境适应性中的作用,利用生物信息学和qRT-PCR技术对其RcCATs和RcSODs基因家族成员的序列和表达模式进行了分析,并构建RcCAT和RcSOD1的pYES2.0表达载体与pGBKT7诱饵载体,分别进行了酵母胁迫分析和拟南芥酵母文库中互作蛋白的筛选。结果显示:(1)大花红景天中共有2条RcCAT基因,3条RcSOD基因和1条Cu/Zn SOD铜伴侣基因RcCCS。生物信息学分析显示上述基因的氨基酸序列与其同源基因具有较高的相似性(66.37%～94.51%),且均没有跨膜结构域,亚细胞位置预测RcCATs位于过氧化物酶体,RcSODs和RcCCS位于细胞质或线粒体。(2)qRT-PCR结果显示6个基因在根、茎、叶三个器官中均有表达且主要在叶片中表达,低温胁迫和植物激素ABA对基因表达量的影响显著。(3)酵母胁迫结果显示异源表达RcCAT和RcSOD1基因的阳性酵母菌株在冷、热、盐、碱、重金属和H₂O₂胁迫下的细胞活力均比pYES2.0空载菌株高。(4)通过酵母双杂交共筛选到4个与RcCAT互作明显的基因AtbHLH121(AT3G19860)、AtCPCK2(AT2G23070)、AtGRP4(AT5G50750)和AtRAPTOR1B(AT3G08850),3个与RcSOD1互作明显的基因AtEMB(AT5G11890)、AtMBP2(AT1G52030)和AtRH8(AT4G00660)。综上结果表明,大花红景天能够通过RcCATs和RcSODs基因广泛参与调控其生长发育、胁迫响应等代谢途径来适应高原恶劣的环境。     

Suppression of Botrytis cinerea sporulation by Clonostachys rosea on rose debris: a valuable component in Botrytis blight management in commercial greenhouses

Botrytis blight, caused by Botrytis cinerea (Bc), is an important disease on roses grown in plastic greenhouses in Brazil. Biocontrol with Clonostachys rosea (Cr) applied to leaves and crop debris to reduce pathogen sporulation can complement other control measures for disease management. Two experiments, each with a rose cultivar, were conducted in a plastic greenhouse. For ‘Red Success,’ four treatments were compared: (1) control; (2) fortnightly sprays of Cr; (3) weekly sprays of mancozeb; and (4) weekly sprays of either Cr or mancozeb to the lower third of the plants and the debris. For ‘Sonia,’ treatment 4 was not included. Samples were taken from debris (leaves and petals) at ten 15-day intervals and plated on PCA medium. Sporulation of fungi and incidence of Botrytis blight on buds were assessed. For both cultivars, C treatments significantly (P=0.05) reduced Bc sporulation. However, disease incidence was not consistently reduced, probably because the applications of C. rosea started when Botrytis blight epidemic was advanced and no sanitation practices were performed on nontreated plots. From the present and previous studies, continuous application of Cr on debris, associated with sanitation practices, has the potential to reduce Bc sporulation and disease incidence in the buds.     

蛇足石杉内生真菌Shiraia sp. Slf14中赖氨酸脱羧酶基因的克隆、表达及功能

【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。