五唇兰雌配子体发育和胚胎发生的研究

五唇兰的胚珠倒生型,具薄珠心,两层珠被。胚囊发育为双孢子葱型,成熟胚囊８核。从传粉到受精约５０ｄ,正常双受精。胚具５－６细胞的胚柄,种子成熟时胚柄及胚乳核消失,成熟种子只具单层细胞的种皮和一个未分化的珠珠形胚。     

Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.     

Disease-driven detection of differential inherited SNP modules from SNP network

Detection of the synergetic effects between variants, such as single-nucleotide polymorphisms (SNPs), is crucial for understanding the genetic characters of complex diseases. Here, we proposed a two-step approach to detect differentially inherited SNP modules (synergetic SNP units) from a SNP network. First, SNP-SNP interactions are identified based on prior biological knowledge, such as their adjacency on the chromosome or degree of relatedness between the functional relationships of their genes. These interactions form SNP networks. Second, disease-risk SNP modules (or sub-networks) are prioritised by their differentially inherited properties in IBD (Identity by Descent) profiles of affected and unaffected sibpairs. The search process is driven by the disease information and follows the structure of a SNP network. Simulation studies have indicated that this approach achieves high accuracy and a low false-positive rate in the identification of known disease-susceptible SNPs. Applying this method to an alcoholism dataset, we found that flexible patterns of susceptible SNP combinations do play a role in complex diseases, and some known genes were detected through these risk SNP modules. One example is GRM7, a known alcoholism gene successfully detected by a SNP module comprised of two SNPs, but neither of the two SNPs was significantly associated with the disease in single-locus analysis. These identified genes are also enriched in some pathways associated with alcoholism, including the calcium signalling pathway, axon guidance and neuroactive ligand-receptor interaction. The integration of network biology and genetic analysis provides putative functional bridges between genetic variants and candidate genes or pathways, thereby providing new insight into the aetiology of complex diseases.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

不同培养条件对猪卵母细胞IVM、IVF的影响

通过优化猪卵母细胞体外成熟、体外受精和胚胎体外发育体系,以进一步提高体外胚胎的生产效率和质量.研究了激素存在时间、不同激素和不同血清对猪卵母细胞体外成熟的影响;共培养体系、精卵作用时间、去除卵丘细胞的方法对猪体外受精及早期胚胎发育的影响.猪卵母细胞IVM培养48h,前24h内加入PMSG、hCG,后24h将其去除,卵母细胞总成熟率为79.54%;培养液添加15?S或15%NCS,卵母细胞成熟率分别为79.48%和74.81%;PMSG、HCG和E2配合使用后卵母细胞成熟率为81.42%.在IVF前用吹打法获得的卵裂率、桑椹胚率分别为37.89%和8.54%,精卵共孵育6h或8h的卵裂率(40.52%,37.24%)、桑椹胚率(8.42%,7.85%),以及用输卵管上皮细胞共培养所获得的卵裂率(40.84%)、桑椹胚率(9.53%)均显著高于其它各组.     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

SpltMNPV日本分离株gp41的克隆表达及gp41和ph的进化分析

从斜纹夜蛾核型多角体病毒(Spodopteralitura multicapsid nucleopolyhedrovirus,SpltMNPV)日本分离株(C3)基因组中克隆了gp41基因.该基因编码区含993bp核苷酸,编码分子量为36.9kDa的多肽.将该基因克隆至原核表达载体pET28a,经IPTG诱导后在大肠杆菌BL21(DE3)中获得了表达.应用CLUSTAL程序分析表明,SpltMNPV日本株(C3)gp41的核苷酸序列和氨基酸序列与SpltMNPV中国G2株相似性最高,均达99.9%.用MEGA分别构建了基于gp41和ph的聚类分析图和分子进化树,发现它们具有相似的拓扑结构.将这两个基因序列结合在一起构建进化树,该树的结构与基于gp41的进化树相似.突变率分析显示gp41的突变率高于ph,这意味着在杆状病毒进化过程中,gp41和ph面临不同的选择压力.     

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.