Identification of the gene MdSOD3 and its role in resisting heavy metal stress in housefly (Musca domestica)

超氧化物歧化酶(SOD)是一种重要的抗氧化酶,在昆虫抗氧化保护过程中起着重要作用.本研究通过RTPCR技术鉴定了家蝇Musca domestica MdSOD3基因(GenBank登录号为JQ408979),cDNA序列817 bp,开放阅读框534 bp,推导的多肽序列含有177个氨基酸.同源性分析及NJ法系统分析表明MdSOD3与其他物种的Cu/ZnSOD关系较近.荧光定量PCR检测该基因在家蝇幼虫脂肪体、肠、表皮和血细胞中不存在差异表达;在受到不同浓度重金属Cd2+刺激时,MdSOD3基因呈诱导性表达,5 mmol/L时表达量最高.通过RNAi策略技术成功敲低MdSOD3表达水平.将RNA干扰60h的幼虫置于5 mmol/L Cd2+处理24h后死亡率明显升高,并且出现中毒表象.NBT活性染色检测到体外重组表达的MdSOD3具有明显的酶活性.结果提示MdSOD3基因可能在家蝇抗逆防御过程中起着重要作用.     

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.     

Comparative study of myocytes from normal and mdx mice iPS cells

Recently, induced pluripotent stem cells (iPS cells) have been derived from various techniques and show great potential for therapy of human diseases. Furthermore, the iPS technique can be used to provide cell models to explore pathological mechanisms of many human diseases in vitro, such as Duchenne muscular dystrophy (DMD), which is a severe recessive X-linked form of muscular dystrophy without effective treatment. In this study, we try to determine whether there are different characteristics of myocytes from mdx iPS cells and C57BL/10 iPS cells. Our results showed that both of mdx and C57BL/10 cells could be induced into iPS cells in vitro, whereas colony-forming ability of mdx iPS cells was much weaker than that of C57BL/10 iPS cells. Meanwhile, mdx iPS cells could be induced to differentiate into myocytes, whereas their differentiation efficiency was much lower than that of C57BL/10 iPS cells. And, the number of apoptotic cells in differentiated myocytes from mdx iPS cells was significantly higher than that from C57BL/10 iPS cells. More importantly, treatment of a pan-caspase inhibitor (Z-VAD) produced a significant decrease in apoptotic cells. This study might add some insight to the biology study of dystrophin gene.     

Construction of a Full-Length cDNA Library and Analysis of Expressed Sequence Tags from Inflorescence of Apomictic Sabaigrass (Eulaliopsis binata)

Eulaliopsis binata, which is a close relative of cereal crops, was recognized as an important research material owing to its high frequency of apospory and autonomous endosperm formation. However, little information is known about its genomics and regulatory pathway participating in reproductive development. For the first step to understand molecular basis in sabaigrass (E. binata), a SMART complementary DNA library from the inflorescence tissue was constructed and characterized. The titers of original and amplified libraries were 5.53 × 10⁶ and 1.49 × 10¹⁰ pfu/ml, respectively. The percentage of recombinants was 96% in the original library. Analysis of sequencing results of 398 out of 437 randomly picked clones showed that 271 (68.1%) expressed sequence tags (ESTs) exhibited significant similarity with known putative functional nucleotide sequences in the GenBank databases, 25 (6.3%) ESTs have significant matches with hypothetical proteins, putative proteins, and unknown proteins, and the other 25.6% ESTs had no significant similarity to sequences in the public databases. Based on molecular function of GO annotation, the four most abundant terms are nucleotide binding, hydrolase activity, ion binding, and protein binding, and these genes were involved in 61 different pathways using the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Besides, simple sequence repeats detection in 398 ESTs was carried out, and several genes were chosen to perform expression analysis. This report represents a first step in expanding molecular-genetic analyses in E. binata and can be used to optimally mine useful information from a relatively small data set.     

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.