Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.     

Development of ELISA for Detection of Mercury Based on Specific Monoclonal Antibodies Against Mercury-Chelate

Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31?×?10⁹ L/mol and its cross-reactivity (CR) with other metals was <2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg²⁺ measurements. The detection range was 0.087–790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.     

Enrichment of a common wheat genetic map and QTL mapping for fatty acid content in grain

DArT and SSR markers were used to saturate and improve a previous genetic map of RILs derived from the cross Chuan35050 × Shannong483. The new map comprised 719 loci, 561 of which were located on specific chromosomes, giving a total map length of 4008.4 cM; the rest 158 loci were mapped to the most likely intervals. The average chromosome length was 190.9 cM and the marker density was 7.15 cM per marker interval. Among the 719 loci, the majority of marker loci were DArTs (361); the rest included 170 SSRs, 100 EST-SSRs, and 88 other molecular and biochemical loci. QTL mapping for fatty acid content in wheat grain was conducted in this study. Forty QTLs were detected in different environments, with single QTL explaining 3.6-58.1% of the phenotypic variations. These QTLs were distributed on 16 chromosomes. Twenty-two QTLs showed positive additive effects, with Chuan35050 increasing the QTL effects, whereas 18 QTLs were negative with increasing effects from Shannong483. Six sets of co-located QTLs for different traits occurred on chromosomes 1B, 1D, 2D, 5D, and 6B.     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.     

Survival and differentiation of mammary epithelial cells in mammary gland development require nuclear retention of Id2 due to RANK signaling

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.     

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.     

Atg3-mediated lipidation of Atg8 is involved in encystation of Acanthamoeba

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.