Molecular characterization and evaluation of mycorrhizal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants

Suillus fungal specimens of pine forests from a Mediterranean area of central Spain (Madrid region) were studied based on molecular and physiological analysis of sporocarps to obtain fungal native inocula to produce mycorrhizal Pinus halepensis Miller in nursery. Variation within the internal transcribed spacer (ITS) region of the ribosomal RNA genes of Suillus was examined by restriction fragment length polymorphism (RFLP) and direct sequencing of polymerase chain reaction products. Ribosomal DNA (rDNA) spacers were amplified from pure cultures obtained from fruit bodies of a range of Suillus species: Suillus bellinii (Inzenga) Watling, Suillus bovinus (Pers.) Kuntze, Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.) Snell, Suillus mediterraneensis (Jacquet. & Blum) Redeuil, Suillus luteus L. (Gray), and Suillus variegatus (Sw.) Kuntze. Interspecific variation in the length and number of restriction sites of the amplified ITS region was observed. This variation was confirmed by sequencing, which allowed us to identify some isolates. This is the first time that the ITS sequence of S. mediterraneensis is completely described. No intraspecific rDNA variation was observed within isolates of S. collinitus, S. mediterraneensis, and S. luteus. The phylogenetic analysis established the close relationship among these Mediterranean fungal species. As a further step to characterize the different isolates and to understand the relation between genetic and functional diversity, some physiological variables were evaluated. Intraspecific variation in axenic fungal growth and in mycorrhizal capacities was detected, especially within S. collinitus isolates. The fungal isolates stimulated the growth of P. halepensis in different rates. These studies indicated that ITS analysis, in conjunction with mycorrhizal tests, provides suitable combined tools for the analysis of Suillus spp. in a small geographic area for selecting isolates with final afforestation purposes.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze]

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clonedr1 (droughtresponsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clonedr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clonedr3, responded similarly to clonedr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters     

Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze

Rosmarinic acid, an important phenolic active compound, is one of the main active constituents of Agastache rugosa Kuntze and has astringent properties, antioxidant capacity, anti-inflammatory activity, antimutagenic ability, antimicrobial capacity, and antiviral properties. To investigate in vitro production of rosmarinic acid, we established a hairy root culture of A. rugosa by infecting leaf and stem explants with Agrobacterium rhizogenes R1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (14.1 g dry wt/l) was attained after 14 days of culture, at which time the content of rosmarinic acid was 116 mg/g dry wt. The present results demonstrate that hairy root culture of A. rugosa is a valuable alternative approach for the production of rosmarinic acid.     

Isolation and characterization of 11 microsatellite loci from Camellia sinensis in Taiwan using PCR-based isolation of microsatellite arrays (PIMA)

We report 11 novel microsatellite primer pairs for the wild tea, Camellia sinensis (L.) O. Kuntze forma formosensis Kitamura. These simple sequence repeat markers were tested in 24 samples collected from wild tea populations, and in cultivars and C. japonica. The number of alleles ranged from 4 to18. The expected (H _E) and observed (H _O) heterozygosity were 0.687–0.946 and 0.042–0.792, respectively. All loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the rare species. Significant LD was discovered in most loci. These primers may provide a tool for understanding demography and population structure in wild tea.     

新疆野苹果AP2/ERF转录因子家族鉴定与响应腐烂病的表达分析北大核心CSCD

AP2/ERF转录因子家族参与了植物生长发育、抵抗胁迫以及植物激素响应等诸多生物过程,是植物中最重要的转录因子家族之一。该研究基于腐烂病菌侵染后的新疆野苹果(Malus sieversii)全长转录组,使用AP2保守结构域的隐马可夫模型PF00847,鉴定新疆野苹果的AP2/ERF家族成员。利用MEGA6、NCBI CDD-batch、MEME、EXPASY、BUSCA对MsAP2/ERF家族成员进行鉴定、分类和结构分析、理化性质和亚细胞定位分析。通过RNA-seq数据和qRT-PCR实验对差异表达的MsAP2/ERFs基因的表达水平进行分析和验证,旨在鉴定新疆野苹果中潜在具有腐烂病抗性的AP2/ERF家族基因资源。结果显示:(1)在新疆野苹果中共鉴定获得106个AP2/ERF基因,涵盖全部AP2(17个)、ERF(57个)、DREB(25个)、RAV(5个)和Soloist(2个)5个亚家族。(2)进一步的细化分类发现MsERF亚家族包括B1-B6 6个组,而MsDREB亚家族中只有A2、A4、A5、A6共4个组,缺少A1和A3组的基因成员。(3)RNA-seq表达模式分析结果表明,29个MsAP2/ERF基因在腐烂病感染过程中差异表达,其中MsERF亚家族中差异表达基因数量最多(19个)。(4)12个MsAP2/ERF代表基因的qRT-PCR结果表明:8个ERF亚家族基因均受腐烂病病菌诱导显著上调表达,其中B4类ERF成员基因(MsERF40)在腐烂病病菌侵染后5 d表达量上调表达倍数最高;4个MsDREB基因中,3个受到腐烂病病原菌诱导显著上调,1个下调表达;此外,含有TIR保守结构域的MsERF05在腐烂病病菌侵染1 d后上调表达69倍,表明ERF亚家族的MsERF40和MsERF05在新疆野苹果抗腐烂病过程中发挥重要作用。该研究结果为新疆野苹果AP2/ERF基因响应腐烂病的功能和机理研究奠定了基础。     

靖西铁线莲, 广西毛茛科一新种

该文描述了自广西发现的毛茛科铁线莲一新种,靖西铁线莲,此新种与宝岛铁线莲在亲缘关系上接近,与后者的区别在于本种的茎疏被短柔毛,小叶卵形或狭卵形,不分裂,聚伞花序有1～3花,萼片较大长1.7～2 cm,宽0.5～0.7 cm。     

Materials for the fungus flora of Japan (51)

A discomycete in Sarcoscyphaceae is described and illustrated as new to Japan:Cookeina sulcipes (Berk.) Kuntze, found on decayed wood along the Aira River, Iriomote Island, Okinawa Pref., Japan. Hongo, T., Nippon Kingakukai Kaiho (Trans. Mycol. Soc. Japan)38: 99–100, 1997.     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

Prevalence of Helicobacter pylori genotypes (vacA, cagA, cagE and virB11) in gastric cancer in Brazilian's patients: an association with histopathological parameters

Purpose: To investigate the frequency and the association of vacA alleles, cagA, cagE and virB11 genes of Helicobacter pylori from patients with gastric cancer, considering the clinic histopathological parameters. Methods: One hundred and one gastric adenocarcinoma tissues were assessed by PCR to detect H. pylori and vacA alleles, cagA, cagE and virB11. Results: The distribution of cases according to the presence of the genes studied showed that the group containing vacA s1m1, cagA, cagE and virB11 H. pylori genes was significantly more frequent, followed by the group with at least one marker on the right side and left of the island. They were also present in the early stages and were the most frequent in nearly all histopathological grades. Conclusions: This study verified that vacAs1m1 and cag-PAI genes, cagA, cagE and virB11 are important H. pylori markers for gastric cancer development. Also, this study corroborates the importance of cagE and cagA together as cag-PAI marker.     

3种菊科入侵植物不同生长时期的土壤酶活性和养分变化

【目的】黄顶菊、三叶鬼针草和豚草是我国危害较严重的3种菊科入侵植物。了解3种菊科植物生长过程中土壤养分和酶活性的变化,可以为研究其入侵机制提供依据。【方法】在中国农业科学院植物保护研究所廊坊中试基地开展同质园实验,比较分析了黄顶菊、三叶鬼针草和豚草3种菊科入侵植物在幼苗期、旺盛生长期和生殖生长期根际土壤养分和酶活性的变化。【结果】3种外来菊科植物的生长时期对土壤养分和酶活性存在显著影响。3种外来植物入侵域的土壤速效磷、硝态氮含量以及磷酸酶活性随生长时期变化都表现出先升高后降低的趋势,均在旺盛生长期达到最大值。入侵域土壤速效磷和硝态氮含量的变化趋势与狗尾草明显不同。3种外来植物入侵降低了土壤速效钾含量,提高了土壤脲酶、磷酸酶活性。【结论】3种外来菊科植物的入侵改变了土壤养分和酶活性,创造出更有利于自身生长和繁殖的条件,以利于其进一步扩张。     

Implications of <Emphasis Type="Italic">XRCC1</Emphasis>, <Emphasis Type="Italic">XPD</Emphasis> and <Emphasis Type="Italic">APE1</Emphasis> gene polymorphism in North Indian population: a comparative approach in different ethnic groups worldwide

Identifying risk factors for human cancers should consider combinations of genetic variations and environmental exposures. Several polymorphisms in DNA repair genes have impact on repair and cancer susceptibility. We focused on X-ray repair cross-complementing group 1 (XRCC1), Xeroderma pigmentosum D (XPD) and apurinic/apyrimidinic endonuclease (APE1) as these are most extensively studied in cancer. Present study was conducted to determine distribution of XRCC1 C26304T, G27466A, G23591A, APE1 T2197G and XPD A35931C gene polymorphisms in North Indian population and compare with different populations globally. PCR-based analysis was conducted in 209 normal healthy individuals of similar ethnicity. Allelic frequencies in wild type of XRCC1 C26304T were 91.1% C(Arg); G27466A 62.9% G(Arg); G23591A 60.3% G(Arg); APE1 T2197G 75.1% T(Asp) and XPD A35931C 71.8% A(Lys). The variant allele frequency were 8.9% T(Trp) in XRCC1 C26304T; 37.1% A(His) in G27466A; 39.7% A(Gln) in G23591A; 24.9% G(Glu) in APE1 and 28.2% C(Gln) in XPD respectively. We further compared frequency distribution for these genes with various published studies in different ethnicity. Our results suggest that frequency in these DNA repair genes exhibit distinctive pattern in India that could be attributed to ethnicity variation. This could assist in high-risk screening of humans exposed to environmental carcinogens and cancer predisposition in different ethnic groups.     

分离自苏云金芽胞杆菌的首例细菌乌头酸异构酶TbrA的酶学性质研究

[目的]乌头酸异构酶(aconitate isomerase,AI)可介导具有多重生物学活性及应用潜力的小分子物质反式乌头酸(trans-aconitic acid,TAA)的合成.本文通过表征来自苏云金芽胞杆菌的生物体首条AI基因(tbrA)的产物——TbrA蛋白的催化性质,填补人们对于AI酶学特性的认识.[方法]我...     

植物替代控制3种入侵杂草技术的研究与应用进展

植物替代控制是利用一种或多种植物的生长优势控制入侵杂草的方法,它是控制外来杂草危害的有效途径之一;因其既可控制入侵杂草危害又能取得一定的经济效益及生态效益而被人们广泛接受。本文简要介绍了我国3种入侵杂草植物替代控制技术研究与应用现状,系统总结了植物替代控制技术阻截和修复豚草、紫茎泽兰及黄顶菊扩散危害的技术模式和效果,提出了植物替代控制技术未来研究的方向和重点。     

Microarray-based global differential expression profiling of P450 monooxygenases and regulatory proteins for signal transduction pathways in the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Whole genome sequencing of the model white rot basidiomycete Phanerochaete chrysosporium has revealed the largest P450 contingent known to date in fungi, along with related phase I and phase II metabolic genes and signaling cascade genes. As a part of their functional characterization, genome-wide expression profiling under physiologically distinct conditions, nutrient-limited (ligninolytic) and nutrient-rich (non-ligninolytic), was investigated using a custom-designed 70-mer oligonucleotide microarray developed based on 190 target genes and 23 control genes. All 150 P450 genes were found to be expressible under the test conditions, with 27 genes showing differential expression based on a >twofold arbitrary cut-off limit. Of these, 23 P450 genes were upregulated (twofold to ninefold) in defined high-nitrogen cultures whereas four genes were upregulated (twofold to twentyfold) in defined low-nitrogen cultures. Furthermore, tandem P450 member genes in ten of the 16 P450 genomic clusters showed nonassortative regulation of expression reflecting their functional diversity. Full-length cDNAs for two of the high-nitrogen upregulated genes pc-hn1 (CYP5035A1) and pc-hn2 (CYP5036A1) and partial cDNA for a low-nitrogen upregulated gene pc-ln1 (CYP5037A1) were cloned and characterized. The study provided first molecular evidence for the presence of active components of the cAMP- and MAP kinase-signaling pathways in a white rot fungus; four of these components (cpka and ste-12 of cAMP pathway and two MAP kinases, mps1 and sps1) were significantly upregulated (fourfold to eightfold) under nutrient-limited conditions, implying their likely role in the regulation of gene expression involved in secondary metabolism and biodegradation processes under these conditions.