稻田生态系统中3种蜘蛛对白背飞虱的捕食效应研究

应用 二次正交旋转组合设计方法,对稻田生态系统中３种捕食性天敌（三突花蛛Ｍｉｓｕｍｅｎｏｐｓ ｔｒｉｃｕｓｐｉｄａｔａ（Ｔａｂｒｉｃｉｕｓ）、草间钻头蛛Ｈｙｌｙｐｈａｎｔｅｓ ｇｒａｍｉｎｉｃｏｒａ（Ｓｕｎｄｅｖａｌｌ）、真水狼蛛Ｐｉｒａｔａ ｐｉｒａｔｉｃｕｓ（Ｃｌｅｒｋ）和１种害虫（白背飞虱Ｓｏｇａｔｅｌｌａ ｆｕｒｃｉｆｅｒａ（Ｈｏｒｖａｔｈ）的共存系统进行研究,获得了天敌对害虫控制作用的数     

冷藏对美洲斑潜蝇蛹期发育和生存的影响

【目的】美洲斑潜蝇Liriomyza sativae Blanchard自20世纪90年代入侵我国后已经成为蔬菜和观赏植物的重要害虫。然而,近些年该昆虫已经逐渐成为研究植物和昆虫、昆虫抗寒性和寄主适应性等热点课题的模式种类。因此,为了实验室条件下更好保藏该虫的种群,本文研究了冷藏(世代发育起点温度8℃)对该斑潜蝇蛹发育和生存的影响。【方法】以化蛹后第3天的蛹为实验对象,8℃低温冷藏,并以冷藏时间(0、7、14、21、28和35 d)为参试因子研究了冷藏时间对美洲斑潜蝇蛹羽化率的影响;化蛹后每24 h解剖观察一次蛹并对蛹期发育阶段进行划分;根据划分的蛹期阶段,对处理中未羽化的蛹进行解剖比对,得到对低温最敏感的蛹期。【结果】随着冷藏时间的增长,美洲斑潜蝇的蛹羽化率逐渐降低。超过14 d后,羽化率明显下降。冷藏至35 d时羽化率为零。解剖正常发育的美洲斑潜蝇的蛹,将蛹期分为预蛹期、隐头蛹期、显头蛹初期、半透明眼期、浅黄色到琥珀色眼期、红褐色眼期、鬃毛蛹期。不同冷藏时间条件下,未羽化的蛹死亡时间主要为鬃毛蛹期,且鬃毛蛹期死亡的蛹的数量与贮藏时间呈正比例关系。【结论】在室内冷藏美洲斑潜蝇时,3日龄蛹8℃贮藏少于14 d为宜;鬃毛蛹期(10~11日龄蛹)对低温比较敏感,冷藏过程中,应避免鬃毛蛹期的蛹。     

Methyl-CpG-Binding Domain Protein MBD7 Is Required for Active DNA Demethylation in Arabidopsis

Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis.DNA methylation is an important epigenetic marker for genome stability and the regulation of gene expression in both plants and animals (Law and Jacobsen, 2010; He et al., 2011). In plants, the molecular mechanisms for DNA methylation have been well characterized by the use of powerful genetic screening systems (Bartee et al., 2001; Lindroth et al., 2001; Matzke et al., 2004; He et al., 2009). A transgene or an endogenous gene may be silenced because of DNA hypermethylation in the promoter region. Screenings for mutants with release of the silenced marker genes have identified many components that are involved in RNA-directed DNA methylation (RdDM) and in maintaining DNA methylation (Matzke and Birchler, 2005; Law and Jacobsen, 2009; He et al., 2011; Bender, 2012). DNA methylation is catalyzed by DNA methyltransferases including DNA METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3), which maintain symmetric CG and CHG methylation, respectively, during DNA replication, and DOMAINS REARRANGED METHYLASE2 (DRM2) and CMT2, which are required for establishing CHG and asymmetric CHH methylation during each cell cycle. DRM2 also catalyzes CG methylation (Law and Jacobsen, 2010; Haag and Pikaard, 2011; He et al., 2011; Zemach et al., 2013; Stroud et al., 2014). Twenty-four-nucleotide small RNAs produced through the RdDM pathway target genomic regions to guide the establishment of DNA methylation by DRM2 (Cao et al., 2003).DNA methylation can be actively removed by a subfamily of bifunctional DNA glycosylases/lyases including REPRESSOR OF SILENCING1 (ROS1; Gong et al., 2002) and its paralogs DEMETER and DEMETER-LIKE2/3 (Gehring et al., 2006; Ortega-Galisteo et al., 2008). DNA methylation can also be passively lost during DNA replication when DNA methylation cannot be maintained (Zhu, 2009). Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) in the ProRD29A:LUC/Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenic Arabidopsis (Arabidopsis thaliana) line has been used as a marker to identify ros1 and ros3 mutants in which both ProRD29A:LUC and Pro35S:NPTII are silenced (Gong et al., 2002; Zheng et al., 2008). ROS3 is an RNA-binding protein that facilitates the function of ROS1 in active DNA demethylation at certain genomic loci. Using Pro35S:NPTII as a selection marker for kanamycin-sensitive mutants and the 35S-SUC2 transgene or a chop PCR marker for assaying DNA methylation at the 3′ region of At1g26400 from transfer DNA (T-DNA) insertion mutants, researchers recently identified two genes involved in active DNA demethylation: ROS4/INCREASED DNA METHYLATION1 (IDM1) and ROS5/IDM2 (Li et al., 2012; Qian et al., 2012, 2014; Zhao et al., 2014). ROS4/IDM1 is a plant homeodomain-finger domain-containing histone acetyltransferase that catalyzes histone H3 lysine18 (H3K18) and lysine23 (H3K23) acetylation (Li et al., 2012; Qian et al., 2012). ROS5/IDM2 is a member of the small heat shock protein family that interacts physically with ROS4/IDM1 for the regulation of active DNA demethylation. Genetic analysis indicates that ROS1, ROS4/IDM1, and ROS5/IDM2 are in the same genetic pathway and that ROS4/IDM1 and ROS5/IDM2 may form a protein complex for the regulation of active DNA demethylation (Qian et al., 2014; Zhao et al., 2014).During the genetic screening for kanamycin-sensitive mutants using the ProRD29A:LUC/Pro35S:NPTII transgenic line in this study, we identified another mutant, mbd7, where the Pro35S:NPTII transgene is specifically silenced. MBD7 is a methyl-CpG-binding domain (MBD) protein containing three MBD motifs that bind in vitro to methylated symmetric CG sites. MBD7 localizes to all highly CpG-methylated chromocenters in vivo (Zemach and Grafi, 2003; Zemach et al., 2008). Recruitment of MBD7 to chromocenters is disrupted in decrease in DNA methylation1 (ddm1) and met1, two mutants with great reductions in DNA methylation, suggesting that DNA methylation is required for proper MBD7 localization (Zemach et al., 2005). In this study, we found that MBD7 interacts physically with ROS5/IDM2 and is required for the active DNA demethylation of certain genomic loci, especially for the Gypsy-type long terminal repeat (LTR) retrotransposons with high densities of DNA methylation around chromocenters in Arabidopsis.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

In vivo specific delivery of c-Met siRNA to glioblastoma using cationic solid lipid nanoparticles

RNA interference is a powerful strategy that inhibits gene expression through specific mRNA degradation. In vivo, however, the application of small interfering RNAs (siRNAs) is severely limited by their instability and their poor delivery into target cells and tissues. This is especially true with glioblastomas (GBMs), the most frequent and malignant form of brain tumor, that has limited treatment options due to the largely impenetrable blood-brain barrier. Here, cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, was conjugated to PEGylated c-Met siRNA. The c-Met siRNA-PEG/SLN complex efficiently down-regulated c-Met expression level, as well as decreased cell proliferation in U-87MG in vitro. In orthotopic U-87MG xenograft tumor model, intravenous administration of the complex significantly inhibited c-Met expression at the tumor tissue and suppressed tumor growth without showing any systemic toxicity in mice. Use of Cy5.5 conjugated SLN revealed enhanced accumulation of the siRNA-PEG/SLN complexes specifically in the brain tumor. Our data demonstrates the feasibility of using siRNA-PEG/SLN complexes as a potential carrier of therapeutic siRNAs for the systemic treatment of GBM in the clinic.     

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.     

麦红吸浆虫唾腺EST-SSRs的信息分析及分子标记筛选

昆虫EST资源库的扩充为开发新的分子标记提供了宝贵的资源。本研究对NCBI的EST数据库中来源于麦红吸浆虫Sitodiplosis mosellana唾腺的1 217条EST序列进行了unigene组装、 SSR信息分析和EST-SSR分子标记筛选。结果表明： 在1 047个unigenes中共找到141个SSR位点, 分布于106个（10.12%）unigenes中, 平均每3.49 kb出现一个SSR位点。在1~6碱基重复基元中, 1~3碱基是主要重复类型, 占总SSR的97.16%以上。A/T（31.21%）, AC/GT（15.60%）和AAC/GTT（9.22%）分别是单、 双和三碱基中占优势的重复基元类型。利用Primer Premier 5.0软件对查找的EST SSRs进行引物设计, 并以麦红吸浆虫基因组DNA为模板, 对从中选出的26对SSR引物进行多态性检测。结果有20对（76.92%）引物能扩增出清晰的目的条带, 并且其中9对（45%）引物表现出多态性。多态性分析结果表明, 从9对EST-SSR引物中, 共检测到51个等位基因, 平均每个位点含有等位基因数为5.67, 平均期望杂合度为0.65, 平均多态信息含量为0.60。本研究能够为今后麦红吸浆虫的种群遗传结构与遗传多样性研究提供帮助。