Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Isolation and Expression Analysis of Two Genes Encoding Cinnamate 4-Hydroxylase from Cotton(Gossypium hirsutum)

Two genes （GhC4H1 and GhC4H2） that encode putative cotton cinnamate 4-hydroxylases that catalyze the second step in the phenylpropanoid pathway were isolated from developing cotton fibers. GhC4H1 and GhC4H2 each contain open reading frames of 1 518 base pairs （bp） in length and both encode proteins consisting of 505 amino acid residues. They are 90.89% identical to each other at the amino acid sequence level and belong to class I of plant C4Hs. GhC4H1 and GhC4H2 genomic DNA are 2 247 and 2 161 bp long, respectively, and contain two introns located at conserved positions relative to the coding sequence. GhC4HI and GhC4H2 promoters were isolated and found to contain many cis-elements （boxes P, L and AC-1 element） previously identified in the promoters of other phenylpropanoid pathway genes. Histochemical staining showed GUS expression driven by the GhC4H1 and GhC4H2 promoters in ovules and fibers tissues. GhC4H1 and GhC4H2 were also widely expressed in other cotton tissues. GhC4H2 expression reached its highest level during the elongation stage of fiber development, whereas GhC4H1 expression increased during the secondary wall development period in cotton fibers. Our results contribute to a better understanding of the biochemical role of GhC4H1 and GhC4H2 in cotton fiber development.

Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp,Labeo rohita

Background: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells(SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp(Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.Results: Rohu Nanog(LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetical y, it was closely related to freshwater counterparts. Protein sequence(386 AA of42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis-à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.Conclusion: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.

Differential Regulatory Mechanisms of CBF Regulon Between Nipponbare (Japonica) and 93-11 (Indica) During Cold Acclimation

Nine CBF/DREB1 homologous genes in rice were obtained by BLAST search in the NCBI database, which share conserved amino acid sequences with DREB1 protein in Arabidopsis. Three CBF genes organized in tandem, named OsCBF1, OsCBF2 and OsCBF3, showed a transient induction in the process of cold acclimation, much stronger in indica rice 93-11 compared with japonica rice Nipponbare. The candidate downstream genes OsLIP5 and OsLIP9 were induced in 93-11 but not in Nipponbare. The differential expression of CBF regulon might be caused by polymorphisms within promoter sequences between these two rice varieties. These results could be useful for utilization of CBF/DREB1 genes and illustration of differences in chilling tolerance between indica and japonica rice varieties.

Responses Taken by Silencing of NFkappaB, STAMP1 and STAMP2 Genes and Expression of NFkB, Act-1, p53 and p73 at -/+ TNFalpha Induced LNCaP Cells

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in men in the Western World. The effects of androgens are mediated by the Androgen Receptor (AR). Therefore, studies focus on the identification of AR-regulated genes that are also highly expressed in the prostate. STAMP family genes STAMP1/STEAP2 and STAMP2/STEAP4 have only expressed in androgen receptor-positive cells, the role of AR in STAMP family gene expression is an important question. STEAP (Six Transmembrane Epithelial Antigens of Prostate) is the first characterized prostate enriched six transmembrane genes, expressed in metastatic prostate cancer samples, it is tempting to speculate that STAMP/STEAP family genes may be involved in similar functions with a role for both the normal biology and pathophysiology of the prostate. Using siRNA technology in LNCaP cells expressing STAMP genes per se, an apoptosis panel including pro-apoptotic and/or apoptotic molecules was assayed by RT-PCR. In this research project, the prostate-specific STAMP gene family and its regulatory effects on the nuclear factor kappa B and caspase-related pathways were characterized. Considering that the beta-actin response in the control group was high in the immunolabeling studies, an increase in the induction of Tumor Necrosis Factor (TNF) was detected in the signals received with the vital proteins NFkB and akt, which were silenced by siRNA, which means that STAMP genes potentiate vital proteins.

High-throughput Binary Vectors for Plant Gene Function Analysis

A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing, and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

Characterization of the Tomato Prosystemin Promoter:Organ-specific Expression,Hormone Specificity and Methyl Jasmonate Responsiveness by Deletion Analysis in Transgenic Tobacco Plants

Tomato systemin is a bioactive peptide that regulates the systemic activation of wound-responsive genes. It is released from its 200 amino acid precursor called prosystemin. Initial tissue-localization and hormone-induced expression assays indicated that the tomato prosystemin gene （SIPS） accumulates mainly in floral tissues and in response to exogenous abscisic acid and methyl jasmonate （MeJA） treatments, respectively. Later, the promoter regions of the PS gene in tomato （Solanum lycopersicum L. cv. Castlemart）, pepper （Capsicum annuum） and potato （Solanum tuberosum） were isolated and an in silico analysis of the SIPS promoter revealed an over-representation of stress- and MeJA-responsive motifs. A subsequent 5＇ deletion analysis of the SIPS promoter fused to the/^-glucuronidase reporter （GUS） gene showed that the -221 to ＋40 bp proximal SIPS promoter region was sufficient to direct the stigma, vascular bundle-specific and MeJA-responsive expression of GUS in transgenic tobacco plants. Important vascular.tissue-specific, light- and MeJA-responsive cis-elements were also present in this region. These findings provide relevant information regarding the transcriptional regulation mechanisms of the SIPS promoter operating in transgenic tobacco plants. They also suggest that its Ussue-specificity and inducible nature could have wide applicability in plant biotechnology.