Effect of GbKTN1 from Gossypium barbadense on cell elongation of fission yeast（Schizosaccharomyces pombe）

The GbKTN1 gene was isolated from 10 DPA fiber cells of Gossypium barbadense using 5′RACE/3′RACE.Full-length cDNA of this gene is 2006 bp, including a 113 bp of 5′untranslated region, a 1563 bp of an open reading frame(ORF), and a 327 bp of 3′untranslated region (excluding the stop codon TAA). The ORF of GbKTN1 encodes a 521-amino acid protein with a predicted size of 55 kD. Near C-terminal of the deduced protein there is a putative ATP binding site between amino acid residues from 233 to 414. Southern blot analysis indicated that the GbKTN1 was a single copy gene in G barbadense. Combining semi-quantitative RT-PCR with Southern blot hybridization revealed that GbKTN1 expressed in all the organs detected such as roots, stems, leaves and fibers. However, the mRNA of GbKTN1 was the most abundant in fiber cells, while it was the lowest in leaves. The GbKTN1 cDNA was transformed into S. pombe to verify its function on cell elongation. Results showed that most yeast cells over expressing GbKTN1 gene were elongated dramatically with an average length increase of 2.18 times than that of the non-induced cells. Even the morphology of some yeast cells appeared irregularly. To the best of our knowledge this is the first evidence that KTN1 is correlated with cell elongation in vivo.     

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.     

<Emphasis Type="Italic">Cis</Emphasis>-regulatory change and expression divergence between duplicate genes formed by genome duplication of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

As an index of functional divergence, expression divergence between duplicate gene copies has been observed and correlated with protein coding sequence divergence and bias in gene functional classes. However, the changes in the cis-regulatory region of the duplicate genes which is thought to have important role in expression divergence, has not been explored on the genome-wide scale. We analyzed functional genomics data for a large number of duplicated gene pairs formed by ancient polyploidy events in Arabidopsis thaliana. The divergence in cis-regulatory regions between two copies is positively correlated with the magnitude difference of expression. Moreover, we find that highly expressed duplicate gene pairs have a more diverged cis-regulatory region than weakly expressed gene pairs. We also show that the correlation between expression functional constraint and protein functional constraint is different in old and young duplicate pairs. Our results suggest that cis-regulatory sequence divergence contributes to the expression divergence of duplicate genes formed by genome-wide du-plication. Cis-regulatory region diverges faster in highly expressed duplicate pairs. The diversify selection strengths that act on cis-regulatory region and protein coding region are negatively correlated in young duplicate pairs under expression con-straint.     

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.     

Characterization of a calmodulin binding protein kinase from<Emphasis Type="Italic">Arabidopsis thalian</Emphasis>

A full-length calmodulin binding protein kinase cDNA ,AtCBK1 ,from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE-Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues,The phylogenetic analyses revenl that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup,indicating that they may come from different ancestors,The result suggests that AtCBK1 encoldes a CaM-binding serine/threonine protein kinase.     

Analysis of type II toxin-antitoxin genes <Emphasis Type="Italic">all</Emphasis> 3211-<Emphasis Type="Italic">asl</Emphasis> 3212 in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

The type II toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses, a similar function to heterocyst of cyanobacteria. In this paper, we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type II toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to MazF, a member of mazEF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity, the sensitivity to antibiotics, and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus, we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.     

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

Mapping of genetic modifiers of <Emphasis Type="Italic">Plcd1</Emphasis> in scant hair mice (<Emphasis Type="Italic">snthr</Emphasis><Superscript><Emphasis Type="Italic">1Bao</Emphasis></Superscript>)

The scant hair mutant mouse (locus symbol: snthr ^1Bao ) is a recessive mutation that originated in an ethylnitrosourea chemical carcinogenesis study using the DBA/2J inbred strain. The gene responsible for the mutation was previously determined to be phospholipase C, delta 1 (Plcd1; mutant allele symbol Plcd1 ^snthr1Bao ). To map the modifiers of Plcd1, an intercross (DBA/2J-snthr ^1Bao /snthr ^1Bao × C57BL/6J+/+) was conducted. The F2 mutant progeny exhibited a variety of alopecia phenotypes; all F2 mutants (n=507) were classified into 3 groups (mild, moderate, and severe alopecia) and genotyped based on 96 microsatellites. A major QTL was identified on mouse chromosome (mChr) 15 at 12 cM with an LOD score greater than 7 (P < 0.0001). Three minor QTLs were detected on mChr 2, 5, and 7 at 40, 84 and 48 cM, respectively. The QTLs on mChr 7 and 15 were associated with minor alopecia while the QTLs on mChr 2 and 5 were associated with moderate to severe alopecia. No antagonistic or synergistic effects among or between the 4 QTLs were found. Integrating the functions of the 4 potential regulatory QTLs and mutant Plcd1 ^snthr1Bao , we found that these QTLs might contribute to variations of scant hair severity by altering the Ca²⁺ signal pathways in mouse skin.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

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.     

Comparative proteomics analysis of <Emphasis Type="Italic">OsNAS1</Emphasis> transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> under salt stress

Salt stress is one of the major abiotic stresses in agricultural plants worldwide. We used proteomics to analyze the differential expression of proteins in transgenic OsNAS1 and non-transformant Brassica napus treated with 20 mmol/L Na₂CO₃. Total protein from the leaves was extracted and separated through a high-resolution and highly repetitive two-dimensional electrophoresis (2-DE) technology system. Twelve protein spots were reproducibly observed to be upregulated by more than 2-fold between transgenic and non-transformant B. napus. These 12 spots were digested in-gel with trypsin and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain the peptide mass fingerprints. Protein database searching revealed that 5 of these proteins are involved in salt tolerance: dehydrogenase, glutathione S-transferase, peroxidase, 20S proteasome beta subunit, and ribulose-1,5-bisphosphate carboxylase/oxygenase. The potential functions of these identified proteins in substance and energy metabolism, stress tolerance, protein degradation, and cell defense are discussed. The salt tolerance of the transgenic rapeseed was significantly improved by the introduction of the OsNAS1 gene from Brazilian upland rice of Oryza sativa (cv. IAPAR 9).     

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     

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.     

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC 7120

The fecC gene encoding a putative iron (Ⅲ) dicitrate transporte rwas cloned from nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO3^- , NH1^- or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that the fecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed that fecC gene product is required for optimal growth under iron-deficient conditions in Anabaena sp. PCC 7120.     

Cloning,expression of<Emphasis Type="Italic">Crocus sativus</Emphasis> phytoene desaturase gene and preparation of antiserum against it

A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×10⁵. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012) Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.     

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.