Functional analysis of <Emphasis Type="Italic">PI-</Emphasis>like gene in relation to flower development from bamboo (<Emphasis Type="Italic">Bambusa oldhamii</Emphasis>)

Bamboo flowering owns many unique characteristics and remains a mystery. To investigate the molecular mechanisms underlying flower development in bamboo, a petal-identity gene was identified as a PISTILLATA homologue named BoPI from Bambusa oldhamii (bamboo family). Expression analysis showed that BoPI was highly expressed in flower organs and gradually increased during flower development stage, suggesting that BoPI played an important role in flower development. Ectopic expression of BoPI in Arabidopsis caused conversion of sepals to petals. 35S::BoPI fully rescued the defective petal formation in the pi-1 mutant. BoPI could interact with BoAP3 protein in vitro. These results suggested that BoPI regulated flower development of bamboo in a similar way with PI. Besides flower organs, BoPI was also expressed in leaf and branch, which revealed that BoPI may involve in leaf and branch development. Similar to other MIKC-type gene, BoPI contained the C-terminal sequence but its function was controversial. Ectopic expression of the C-terminal deletion construct (BoPI- ΔC) in Arabidopsis converted sepals to petals; BoPI- ΔC interacted with BoAP3 on yeast two-hybrid assay, just like the full-length construct. The result implied that the C-terminal sequence may not be absolutely required for organ identity function in the context of BoPI.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Characterization and Expression Pattern of a Putative Pectin Methylesterase Gene, <Emphasis Type="Italic">BcMF27</Emphasis>, in <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Pectin methylesterases (PMEs) play an important role in modifying cell wall. PMEs catalyze the de-esterification of pectin, an important compound of cell wall, to affect fertility in plant reproduction. However, little especially molecular mechanism about pectin methylesterase is studied in recent years despite its importance to reproductive development in flower plant. Here the bioinformatics analysis of BcMF27 (Brassica campestris Male Fertility 27) (BRAD: Bra000541 GenBank: KT600012) sequence isolated from Brassica campestris L. ssp. chinensis showed its highly and characteristically conserved structure as a pectin methylesterase. Transient expression analysis in the onion epidermal cells revealed the product of BcMF27 was a transmembrane protein. Real-time RT-PCR and in situ hybridization suggested that BcMF27 was expressed in pollen grain and pollen tube. This study demonstrates that BcMF27 encodes a transmembrane pollen- and pollen tube-specific PME gene, and is also considered to help further understand the biological function of pectin methylesterases and the molecular mechanism of pollen development, pollen tube growth as a genic tool.     

The <Emphasis Type="Italic">angora</Emphasis> gene weakens the effect of interaction of the mutant genes <Emphasis Type="Italic">wellhaarig</Emphasis> and <Emphasis Type="Italic">waved alopecia</Emphasis> in mice

The interaction of the mutant genes wellhaarig (we) and waved alopecia (wal) in mice was earlier demonstrated in our laboratory. The we gene significantly accelerates the appearance of alopecia in double we/wewal/wal homozygotes as compared to that in single +/+wal/wal homozygotes. It has been found in this work that the mutant gene angora-Y (Fgf5 ^go-Y ) weakens the effect of interaction of the we and wal genes. The first signs of alopecia appear in mice of the we/wewal/wal genotype at the age of 14 days, in triple Fgf5 ^go-Y /Fgf5 ^go-Y we/wewal/wal homozygotes alopecia is observed seven days later, i. e., in 21-day-old animals. The progression of alopecia in triple homozygotes is expressed to a lesser degree than in double +/+we/wewal/wal homozygotes. A single dose of the Fgf5 ^go-Y gene also decreases the effect of interaction of the we and wal genes, but less than a double dose of this gene. The first signs of alopecia in mice of the +/Fgf5 ^go-Y we/wewal/wal genotype appear only three days later than in double +/+we/wewal/wal homozygotes. The data obtained demonstrate that the Fgf5 ^go-Y gene is a powerful modifier of mutant genes determining the process of alopecia.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

Functional analysis of an <Emphasis Type="Italic">APETALA1</Emphasis>-like MADS box gene from <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> in regulating floral transition and formation

An Eustoma grandiflorum APETALA1 (EgAP1) gene showing high homology to the SQUA subfamily of MADS-box genes was isolated and characterized. EgAP1, containing a conserved euAP1 motif at the C-terminus, showed high sequence identity to Antirrhinum majus SQUAMOSA in the SQUA subfamily. EgAP1 mRNA was detected in the leaf and expressed significantly higher in young flower buds than in mature flower buds. In flowers, EgAP1 mRNA was strongly detected in sepal, weakly detected in petal and was absent in stamen and carpel. Transgenic Arabidopsis plants ectopically expressing EgAP1 flowered early and produced terminal flowers. In addition, the conversion of petals into stamen-like structures was also observed in 35S::EgAP1 flowers. 35S::EgAP1 was able to complement the ap1 flower defects by restoring the defect for sepal formation and significantly increasing second whorl petal production in Arabidopsis ap1 mutant plants. These results revealed that EgAP1 is the APETALA1 homolog in E. grandiflorum and that the function of EgAP1 is involved in floral induction and flower formation.