Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.     

Bioinformatic analysis of BBTV satellite DNA in Hainan

Banana bunchy top virus (BBTV),family Nanaviridae,genus Babuvirus,is a single stranded DNA virus (ssDNA) that causes banana bunchy top disease (BBTD) in banana plants.It is the most common and most destructive of all viruses in these plants and is widespread throughout the Asia-Pacific region.In this study we isolated,cloned and sequenced a BBTV sample from Hainan Island,China.The results from sequencing and bioinformatics analysis indicate this isolate represents a satellite DNA component with 12 DNA sequences motifs.We also predicted the physical and chemical properties,structure,signal peptide,phosphorylation,secondary structure,tertiary structure and functional domains of its encoding protein,and compare them with the corresponding quantities in the replication initiation protein of BBTV DNA1.     

The role of LXRα in goose primary hepatocyte lipogenesis

In this study, we investigate the role of liver X receptor alpha (LXR alpha) in lipogenesis in geese in order to understand the differences in hepatic steatosis mechanisms between mammals and waterfowl. Primary goose hepatocytes were isolated and treated with the LXR alpha agonist T0901317. Triglyceride (TG) accumulation, acetyl-CoA carboxylase alpha (ACC alpha) and fatty acid synthase (FAS) activities, and gene expression levels of LXR alpha, sterol regulatory element-binding proteins-1 (SREBP-1), FAS, ACC alpha and lipoprotein lipase (LPL) were measured in primary hepatocytes. We found a dose-dependent up-regulation of TG accumulation, ACC, and FAS activities and the mRNA levels of LXR alpha, SREBP-1, FAS, ACC alpha, and LPL genes in the presence of To-901317. We also found that binding of nuclear SREBP-1 to ACC alpha SRE sequence was induced by To-901317 (P < 0.05). In conclusion, LXR alpha is involved in the induction of the lipogenic pathway through activation of SREBP-1 and its target genes in goose primary hepatocytes.     

碱性成纤维细胞生长因子在苜蓿中的表达

为了降低bFGF(basic fibroblast growth factor)的生产成本,结合植物生物反应器的优点,就bFGF在转基因苜蓿中的表达进行了探索.将bFGF插入植物表达载体pBⅡ21中,获得了含有bFGF基因的植物表达pBIcbFGF,再将pBIcbFGF利用冻融法转到农杆菌中.利用农杆菌介导法将基因转化保定苜蓿,转基因苜蓿在TM-1培养基+20 mg/L卡那霉素(Kan)+200 mg/L特美汀(Tim)中诱导分化,在生根培养基中生根,获得再生植株.再生植株通过PCR检测、RT-PCR及Western blot证实外源基因已经在苜蓿中成功表达.获得含有目的蛋白的阳性植株.为苜蓿作为植物生物反应器生产bFGF奠定了理论及技术基础.     

蔷薇科植物体细胞胚胎发生及影响因素研究进展

总结了近30年来蔷薇科植物体细胞胚胎发生及影响因素的研究进展。蔷薇科植物体胚发生多数是直接发生途径和间接发生途径同时存在,但以间接发生途径为主。合子胚作为外植体明显好于营养器官作为外植体。诱导体胚发生的植物生长素类调节剂以NAA、2,4-D为主,细胞分裂素类调节剂以6-BA为主,少数植物种类的体胚诱导需要添加KT。冷处理对蔷薇科植物的体胚分化有效。光照对蔷薇科植物的体胚发生没有显著的影响,有时光照会抑制体胚发生。今后应逐步开展对蔷薇科植物体细胞胚胎发生的生理、生化及分子机理的研究,这在蔷薇科植物的新品种培育、遗传改良、优良单株的离体扩殖等具有重要意义。