Fine mapping of a semidwarf gene sd-g in indica rice（Oryza sativa L.）

The semidwarf gene sd-g which has been usedin indiea rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived fromthe cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region.The sd-g was further mapped between two microsatellitemarkers SSR5-1 and SSR5-51, with genetic distances of 0.1and 0.3 cM, respectively, while cosegregated with SSR418. ABAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloningof the sd-g gene.     

Genetic and physical finemapping of the Sc locus conferring indica-japonica hybrid sterility in rice （Oryza sativa L.）

Hybrid sterility is a major hindrance to utilizing the heterosis in indica-japonica hybrids. To isolate a gene Sc conferring the hybrid sterility, the locus was mapped using molecular markers and an F2 population derived from a cross between near isogenic lines. A primary linkage analysis showed that Sc was linked closely with 4 markers on chromosome 3, on which the genetic distance between a marker RG227 and Sc was 0.07 cM. Chromosome walking with a rice TAC genomic library was carried out using RG227 as a starting probe, and a contig of ca. 320 kb covering the Sc locus was constructed. Two TAC clones, M45EI4 and M90J01 that might cover the Sc locus, were partially sequenced. By searching the rice sequence databases with sequences of the TACs and RG227 a japonica rice BAC sequence, OSJNBb0078P24 was identified. By comparing the TAC and BAC sequences, six new PCR-based markers were developed. With these markers the Sc locus was further mapped to a region of 46 kb. The results suggest that the BAC OSJNBb0078P24 and TAC M45EI4 contain the Sc gene. Six ORFs were predicted in the focused 46-kb region.     

Establishment of widely expressed<Emphasis Type="Italic">hb1f</Emphasis> transgenic mouse lineage and its morphologic analysis

Human nuclear receptor hB1F is a novel member of the fushi tarazu factor Ⅰ subfamily of nuclear receptor superfamily. The studies about its homologous genes indicate that hB1F may play a key role in regulating the metabolic homeostasis of cholesterol. After obtaining the founder mice carrying the trausgeue of hblf by microiujectiou, each founder was mated to normal C57 mouse and the positive F1 by PCR identification of the same founder were iutercrossed within sisters and brothers to establish the trausgeuic mouse lineage. The results of F1, F2 and offspring of test cross identification showed that the widely expressed hb1f trausgeuic mouse lineage was established successfully in this study. The tissue morphology of the trausgeuic lineage was also analyzed preliminarily.     

Southern rice black-streaked dwarf virus: A new proposed <Emphasis Type="Italic">Fijivirus</Emphasis> species in the family <Emphasis Type="Italic">Reoviridae</Emphasis>

For the past several years, a novel dwarf disease has been observed on rice （Oryza sativa） in some regions of Guangdong Province and Hainan Province, southern China. Infected plants showed stunting, dark leaf and small enations on stem and leaf back. Typical Fijivirus viroplasma containing crystalline arrayed spherical virons approximately 70--75 nm in diameter and tubular structures were detected in ultrathin sections by an electron microscope in parenchyma phloem cells of the infected plants. The virus was transmitted to rice seedlings by white-backed planthoppers, Sogatella furcifera （Hemiptera： Delphacidae）, collected in the diseased fields. Analysis of dsRNA extracts from infected plants revealed ten linear segments, which were similar to the electrophoretic profile of Rice black-streaked dwarf virus （RBSDV）. RT-PCR with a single primer which matched to a linker sequence ligated to both 3＇ ends of the viral genomic dsRNAs resulted in amplification of genome segments 9 （S9） and 10 （S10） cDNA products. The complete nucleotide sequences of S9 and S10 were obtained from clones of the RT-PCR amplicon exhibited characteristic properties of Fijivirus including low GC content （34.5% and 35.6%）, genus conserved 5＇ and 3＇ termini sequences and similar genome organization. Blast searches indicated that the sequences of S9 and S10 shared 68.8%--74.9% and 67.1% --77.4% nucleotide identities with those of viruses in the Fijivirus group 2, respectively. These values were similar to those among other viruses in the Fijivirus group 2 and considerably lower than those among RBSDV isolates. Phylogenetic trees based on S9 and S10 nucleotide sequences and their putative amino acid sequences showed that this virus represented a separate branch among other Fijiviruses. The virus was also detected by a nested RT-PCR assay in corn （Zea mays）, barnyard grass （Echinochloa crusgalll）, Juncellus serotinus and flaccidgrass （Pennisetum flaccidum） in and/or adjacent to the infected rice fields. I     

Inducible expression pattern of rice Bowman-Birk inhibitor gene<Emphasis Type="Italic">OsWIP1-2</Emphasis> and its protease inhibitory activity

The WIP1-2 gene was cloned from rice. It be-longs to the Bowman-Birk inhibitor gene family. Northernblot showed that expression of this gene was induced bywounding and jasmonic acid (JA). It indicates that the OsWIPI gene plays an important role in the rice defense sys-tem. The OsWIP1-2 was cloned into pET28a and expressed inE. coli. Its expressed product was purified in the form offusion protein and tested for the inhibitory activities againsttrypsin and chymotrypsin. It was found that the fusion pro-tein could inhibit chymotrypsin, but not trypsin. It was alsofound that the His tag at its C-terminal affected its inhibitoryactivity significantly. The fusion protein with a naturalC-terminal had the inhibitory activity, while no inhibitoryactivity was detected in the fusion protein with a (His)6-tag atits C-terminal. This implies that extra amino acid residues atthe C-terminal of OsWIP1-2 may interfere with its correctfolding. The inhibitory assay indicated that the members ofrice Bowman-Birk inhibitor gene family probably differenti-ated both in their structure and function.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

The VER2 promoter contains repeated sequences and requires vernalization for its activity in winter wheat （Triticum aestivum L.）

Previously, we isolated a vernalization-related gene, VER2, from winter wheat (Triticum aestivum L.) and its expression was restricted in the immature leaves of vernalized wheat seedlings. To further investigate the regulation of VER2 expression and the function of its promoter, we isolated a 41.7 kb genomic clone containing VER2 gene from atransformation-competent artificial chromosome (TAC) library of wheat (Triticum aestivum-Haynaldia villosa). The sequence analysis showed that there were eleven predicted genes in the TAC. The exons of gene 3 corresponded to the cDNA sequence of VER2 gene. Analysis of VER2 promoter structure showed that there were three small repeat sequences divided by two large repeat sequences. The putative response elements, such as abscisic acid response elements (ABRE), MeJA-response elements (Me-JARE), low-temperature response elements (LTR), endosperm expression elements, MYB binding sites and similar elements to GA response elements (GARE), were involved in the VER2 promoter region. Construct containing the VER2 promoter (-5895 to 73) driving GFP reporter gene was bombarded into vernalized or non-verualized immature leaves in wheat. The vernalized immature leaves showed bright green fluorescence after incubation for 24 h, however, the green fluorescence was not observed in the non-vernalization leaves under the same condition. These results suggested that vernalization was essential for the function of VER2 promoter in the immature leaves of winter wheat.     

Transfer of bacterial blight resistance from <Emphasis Type="Italic">Oryza meyeriana</Emphasis> to <Emphasis Type="Italic">O. sativa</Emphasis> L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were produced between cultivated rice (Oryza sativa L.) and wild species [O. meyeriana (Zoll. etMor, exSteud.)] with high resistance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to identify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27--38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Mapping and characterization of a tiller-spreading mutant<Emphasis Type="Italic">lazy-2</Emphasis> in rice

Tiller angle of rice is an important agronomic trait that contributes to breed new varieties with ideal architecture. In this study, we report mapping and characterization of a rice mutant defective in tiller angle. At the seedling stage, the newly developed tillers of the mutant plants grow with a large angle that leads to a “lazy“ phenotype at the mature stage. Genetic analysis indicates that this tillerspreading phenotype is controlled by one recessive gene that is allelic to a reported mutant la. Therefore, the mutant was named la-2 and la renamed la-1. To map and clone LA, we constructed a large mapping population. Genetic linkage analysis showed that the LA gene is located between 2 SSR markers RM202 and RM229. By using the 6 newly-developed molecular markers, the LA gene was placed within a 0.4 cM interval on chromosome 11, allowing us to clone LA and study the mechanism that controls rice tiller angle at the molecular level.     

Fermentation of xylose to produce ethanol by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain containing <Emphasis Type="Italic">XYLA</Emphasis> and <Emphasis Type="Italic">XKS1</Emphasis>

Fermentation of the pentose sugar xylose to produce ethanol using lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyce cerevisise, an efficient ethanol producer, cannot utilize xylose because it lacks the ability to convert xylose to its isomer xylulose. In this study, XYLA gene encoding xylose isomerase (XI) from Thermoanaerobacter tengcongensis MB4T and XKS1 gene encoding xylulokinase (XK) from Pichia stipitis were cloned and functionally coexpressed in Saccharomyces cerevisiae EF-326 to construct a recombinant xylose-utilizing strain. The resulting strain S. cerevisiae EF 1014 not only grew on xylose as sole carbon source, but also produced ethanol under anaerobic conditions. Fermentations performed with different xylose concentrations at different temperatures demonstrated that the highest ethanol productivity was 0.11 g/g xylose when xylose concentration was provided at 50 g/L. Under this condition, 28.4% of xylose was consumed and 1.54 g/L xylitol was formed. An increasing fermentation temperature from 30℃ to 37℃ did not improve ethanol yield.     

Attractiveness of tobacco volatiles induced by <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and <Emphasis Type="Italic">Helicoverpa assulta</Emphasis> to <Emphasis Type="Italic">Campoletis chlorideae</Emphasis>

The attraction of Helicoverpa armigera-and Helicoverpa assulta-induced and mechanical damage-induced tobacco volatiles to Campoletis chlorideae was investigated, and the induced volatiles were analyzed. In windtunnel, C. chlorideae was strongly attracted by herbivoreinduced tobacco volatiles. Mechanically damaged tobacco leaves, whether treated with caterpillar regurgitant or water,were more attractive to the parasitoid than undamaged tobacco leaves. GC-MS analysis revealed that only 4 compounds were released from undamaged tobacco leaves,whereas 13 compounds were commonly emitted from herbivore-infested and mechanically damaged tobacco leaves.Compound β-pinene was specifically induced by the infestation of H. armigera, and (Z)-3-hexenal was only induced by the infestation of H. armigera and H. assulta, whereas hexyl acetate was only induced by mechanical damage. Tobacco leaves infested by H. armigera and H. assulta released larger amounts of volatiles than undamaged tobacco leaves did.Tobacco leaves treated with artificial damage plus caterpillars regurgitant or water emitted the same levels of volatiles,which were higher than that emitted by undamaged tobacco leaves. The emission amounts of single compounds were also different between differently treated plants. The differences were large between herbivore-induced and mechanical damage-induced compounds, and small between H. armigeraand H.assulta-induced compounds, and among compounds emitted from mechanically damaged plants treated with water or caterpillar regurgitant.     

Genetic analysis and gene mapping of a dominant presenescing leaf gene <Emphasis Type="Italic">PSL3</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Leaf senescence as an active process is essential for plant survival and reproduction. However, premature senility is harmful to agricultural production. In this study, a rice mutant, named as psl3 (presescing leaf 3) isolated from EMS-treated Jinhui 10, displays obvious premature senility features both in morphological and physiological level. Genetic analysis showed that mutant trait was controlled by a single dominant gene (PSL3), which was located on rice chromosome 7 between SSR marker c7sr1 and InDel marker ID10 with an interval of 53.5 kb. The result may be useful for the isolation of the PSL3 gene.     

Evolutionary status of<Emphasis Type="Italic">Entamoeba</Emphasis>

In addition to its medical importance as parasitic pathogen, Entamoeba has aroused people‘s interest in its evolutionary status for a long time. Lacking mitochondrion and other intracellular organelles common to typical eukaryotes, Entamoeba and several other amitochondrial protozoans have been recognized as ancient pre-mitochondriate eukaryotes and named “archezoa“, the most primitive extant eukaryotes. It was suggested that they might be living fossils that remained in a primitive stage of evolution before acquisition of organelles, lying close to the transition between prokaryotes and eukaryotes. However, recent studies revealed that Entamoeba contained an organelle, “crypton“ or “mitosome“, which was regarded as specialized or reductive mitochondrion. Relative molecular phylogenetic analyses also indicated the existence or the probable existence of mitochondrion in Entamoeba. Our phylogenetic analysis based on DNA topoisomerase Ⅱ strongly suggested its divergence after some mitchondriate enkaryotes. Here, all these recent researches are reviewed and the evolutionary status of Entamoeba is discussed.     

Biologically active <Emphasis Type="Italic">cis</Emphasis>-cinnamic acid occurs naturally in <Emphasis Type="Italic">Brassica parachinensis</Emphasis>

The biologically active cis-cinnamic acid (cis-CA) has been perceived as a synthetic plant growth regulator for decades,However,in the present study,we found that cis-CA actually exists as a naturally occurring compound in a Brassica plant,This natural growth-regulating substance presents in both the sunlight-irradiated leaf tissue and the non-irradiated root tissue ,The concentrations of cis-CA in both tissues are comparable to the bilogi-cally effective lvels of those major plant hormones,the presence of cis-CA in root tissue suggests that it may be produced through both light-dependent and -independent path-ways or it can be transproted from a plant organ to another.