Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Identification and characterization of rDJL, a novel member of the DnaJ protein family, in rat testis

Applying the method of segmentation of seminiferous tubules combined with DDRT-PCR and cDNA library screening, a novel DnaJ homologue, rDJL was identified in rat testis. The reading frame encodes a protein of 223 amino acid residues containing J domain in the NH2 terminal region. rDJL gene is expressed mainly in testis and rDJL protein was immunolocalized notably in the acrosome region of spermatozoa. Immunoprecipitation experiments showed that rDJL interacted with Hsc70 and clathrin protein. When CHO cells were treated with EGF, rDJL and clathrin protein were found to be colocalized and be concentrated as endosome vesicles. The present findings suggest that rDJL functions as co-chaperone to Hsc70, participates in vesicular trafficking and may play an important role in acrosomogenesis.     

Knockout of frataxin gene causes embryo lethality in Arabidopsis

Frataxin is present in mitochondria of all eukaryotes as well as in the cytoplasm of bacteria. In humans, reduced expression of frataxin is associated with Friedreich's ataxia, a recessive inherited neurodegenerative and cardiac disorder leading to reduced life expectancy. Experimental evidences suggest that frataxin acts as an iron-chaperone protein, donating iron to the proteins involved in [Fe-S] cluster assembly and heme synthesis. It also possibly contributes to the process of iron detoxification and storage. The frataxin homolog from Arabidopsis thaliana (AtFH) is a single nuclear-encoded gene targeted to mitochondria and sharing 65% similarity with animal frataxin. In the present work, we show that the knocking out of AtFH gene causes arrest of Arabidopsis embryo development at the globular stage. Consistently with that, we also show by in situ hybridization that AtFH is expressed, in wt Arabidopsis plants, in ovule primordia as well as in embryos at various stages of development, suggesting a key role of plant frataxin during embryogenesis.     

EST-based identification of immune-relevant genes from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758)

A normalized cDNA library from spleen of Indian catfish, Clarias batrachus, was constructed with a redundancy factor of 2.29. A total of 2045 clones from the library were single-pass sequenced, which generated 1937 high quality ESTs with an average read length of approximately 700 bp. Based on sequence similarities, 65 ESTs were found to be associated with immune functions, which were mainly associated with response to stress, response to chemical stimulus, cellular response to stimulus, response to external stimulus, immune response and regulation of response to stimulus. The immune-relevant gene for CD141, thrombomodulin, has been identified in Teleosts for the first time. Six EST-SSRs and three SNPs were found associated with eight immune-relevant genes. These markers associated with important immune genes would be useful for the identification of trait associated alleles for marker-assisted selection. The identification of the putative immune-related genes provides a meaningful framework to understand the Indian catfish immune system and defense mechanisms.     

Characterization and identification of the integrin family in silkworm, Bombyx mori

As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five β subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins.