马铃薯甲虫结节性硬化复合物基因LdTSC1和LdTSC2基因的克隆及功能分析

【目的】通过RNAi技术明确马铃薯甲虫TOR上游的关键信号集成节点及类胰岛素信号通道下游基因结节性硬化复合物TSC1和TSC2的功能。旨在为探明马铃薯甲虫类胰岛素信号转导提供更多理论支持。【方法】在NCBI(美国国家生物技术信息中心)获取马铃薯甲虫LdTSC1/2序列,分别利用多重序列比对和系统发育分析确定该基因的完整性和系统发育关系;采用喂食幼虫dsRNA的方法,观察该基因的调低对马铃薯甲虫幼虫生长发育、糖脂代谢的影响。【结果】克隆得到马铃薯甲虫TSC1编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁直系同源蛋白的氨基酸序列的自展一致度为100%,聚为一支;TSC2编码蛋白的氨基酸序列与鞘翅目白蜡窄吉丁和赤拟谷盗的同源蛋白氨基酸序列的自展一致度为100%,聚为一支。通过分别喂食2龄幼虫LdTSC1/2的dsRNA能有效降低靶标基因的表达量,幼虫出现体重减轻,化蛹率和羽化率显著下降,葡萄糖的吸收转化效率降低,海藻糖含量升高和甘油三酯均减少。【结论】下调2龄幼虫LdTSC1/2的表达量,导致试虫出现抑制了糖脂代谢、脂肪体减少、体重减轻以及发育延迟;结果表明LdTSC1/2调控了马铃薯甲虫幼虫的糖脂代谢过程,显著影响幼虫化蛹和蜕皮过程。     

Dexmedetomidine alleviates H2O2-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells

Molecular Biology Reports - Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has...     

Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice

Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-1,4-xylanase activities) from mollusca, Ampullaria crossean and a β-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.     

OsDi19‐4 acts downstream of OsCDPK14 to positively regulate ABA response in rice

The drought‐induced 19 protein family consists of several atypical Cys2/His2‐type zinc finger proteins in plants and plays an important role in abiotic stress. In this study, we found that overexpressing OsDi19‐4 in rice altered the expression of a series of abscisic acid (ABA)‐responsive genes, resulting in strong ABA‐hypersensitive phenotypes including ABA‐induced seed germination inhibition, early seedling growth inhibition and stomatal closure. On the contrary, OsDi19‐4 knockdown lines were less sensitive to ABA. Additionally, OsCDPK14 was identified to interact with OsDi19‐4 and be responsible for the phosphorylation of OsDi19‐4, and the phosphorylation of OsDi19‐4 was further enhanced after the treatment of ABA. Apart from these, OsDi19‐4 was shown to directly bind to the promoters of OsASPG1 and OsNAC18 genes, two ABA‐responsive genes, and regulate their expression. Transient expression assays confirmed the direct regulation role of OsDi19‐4, and the regulation was further enhanced by the increased phosphorylation of OsDi19‐4 after the treatment of ABA. Taken together, these data demonstrate that OsDi19‐4 acts downstream of OsCDPK14 to positively regulate ABA response by modulating the expression of ABA‐responsive genes in rice.     

Distribution,Speciation and Bioaccumulation of Hg and As in Mariculture Sediments from Dongshan Bay,China

Hg and As are the major hazardous pollutants in marine sediments due to their high toxicity to benthonic organisms. Understanding the spatial distribution, speciation and bioaccumulation of these toxic elements in sediments is therefore of high environmental importance for identifying their potential risks. Sediments and bivalves Paphia undulata were collected from the mariculture area of Dongshan Bay, China, for characterizing geochemistry (by using the European Community Bureau of Reference (BCR) sequential extraction procedure) and bioaccumulation of Hg and As [by calculating the biota sediment accumulation factor (BSAF)]. Both elements in sediments were mostly associated with the residual fraction (69.52–95.06% and 88.22–91.12% of the total concentration, respectively), followed by the oxidizable (bound to sulfides and organic matter) fraction (1.25–25.32% and 3.62–6.00%, respectively). However, Hg presented a higher bioaccumulation than As. Correlation analysis indicated that As in residual fraction and Hg in oxidizable fraction exert positive contributions (R = 0.927, P < 0.01 and R = 0.869, P < 0.05, respectively) on their own bioaccumulation factor. This indicated that P. undulata could adsorb both Hg in organic fraction and As in residual fraction from the sediments. Therefore, we should pay more attention to the potential dissolution and release of metals bound to sediments in the digestive tracts of marine organisms.     

Characterization of Growth and Reproduction Performance,Transgene Integration,Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.