杜鹃花属植物扦插繁殖研究进展

我国有着丰富的杜鹃花资源,但有关其繁殖应用的研究还具有一定的局限。种子育苗耗时长,组培育苗成本和技术要求高,都不适于杜鹃花属植物的大面积生产。扦插繁殖快,还可保持母本的优良性状。从插条的选择,准备,插条的生根激素处理,扦插基质的选择,外界环境条件对扦插成活率的影响及扦插后的养护管理等六个方面对杜鹃的扦插繁殖技术的研究进行综述,以期推进杜鹃花属植物,尤其是中国野生杜鹃的引种驯化和大面积的推广应用。     

RP-HPLC同时测定喙果绞股蓝中芦丁和槲皮素的含量

目的:建立RP-HPLC同时测定喙果绞股蓝中芦丁和槲皮素含量的方法,并揭示其含量动态变化规律。方法:采用大连依利特SinoChromODS-APC18色谱柱(250 mm×4.6 mm,5μm),流动相:甲醇-0.4%磷酸水溶液梯度洗脱,流速:1 mL/min,检测波长:364 nm,柱温:30℃。结果:芦丁在0.1525～3.8120μg范围内线性关系良好,r=0.9998,平均回收率为98.2%(RSD=2.0%);槲皮素在0.0589～1.4720μg范围内线性关系良好,r=0.9999,平均回收率为97.7%(RSD=2.3%)。含量测定结果表明芦丁和槲皮素的含量具季节性动态变化,芦丁8月份含量高,平均质量分数达6.31 mg/g,槲皮素9月份含量最高,平均质量分数达0.86 mg/g。结论:该方法简单,准确度高,为喙果绞股蓝的质量控制提供实验依据,芦丁和槲皮素含量动态变化规律为其开发利用提供参考。     

Proteomic profiles of mouse neuro N2a cells infected with variant virulence of rabies viruses

We characterized the proteomes of murine N2a cells following infection with three rabies virus (RV) strains, characterized by distinct virulence phenotypes (i.e., virulent BD06, fixed CVS-11, and attenuated SRV9 strains), and identified 35 changes to protein expression using two-dimensional gel electrophoresis in whole-cell lysates. The annotated functions of these proteins are involved in various cytoskeletal, signal transduction, stress response, and metabolic processes. Specifically, a-enolase, prx-4, vimentin, cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and prx-6 were significantly up-regulated, whereas Trx like-1 and galectin-1 were down-regulated following infection of N2a cells with all three rabies virus strains. However, comparing expressions of all 35 proteins affected between BD06-, CVS-11-, and SRV9-infected cells, specific changes in expression were also observed. The up-regulation of vimentin, CIAPIN1, prx-4, and 14-3-3 theta/delta, and downregulation of NDPK-B and HSP-1 with CVS and SRV9 infection were ≥ 2 times greater than with BD06. Meanwhile, Zfp12 protein, splicing factor, and arginine/serine-rich 1 were unaltered in the cells infected with BD06 and CVS- 11, but were up-regulated in the group infected with SRV9. The proteomic alterations described here may suggest that these changes to protein expression correlate with the rabies virus' adaptability and virulence in N2a cells, and hence provides new clues as to the response of N2a host cells to rabies virus infections, and may also aid in uncovering new pathways in these cells that are involved in rabies infections. Further characterization of the functions of the affected proteins may contribute to our understanding of the mechanisms of RV infection and pathogenesis.     

Defining the replication program through the chromatin landscape

DNA replication is an essential cell cycle event required for the accurate and timely duplication of the chromosomes. It is essential that the genome is replicated accurately and completely within the confines of S-phase. Failure to completely copy the genome has the potential to result in catastrophic genomic instability. Replication initiates in a coordinated manner from multiple locations, termed origins of replication, distributed across each of the chromosomes. The selection of these origins of replication is a dynamic process responding to both developmental and tissue-specific signals. In this review, we explore the role of the local chromatin environment in regulating the DNA replication program at the level of origin selection and activation. Finally, there is increasing molecular evidence that the DNA replication program itself affects the chromatin landscape, suggesting that DNA replication is critical for both genetic and epigenetic inheritance.     

The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.     

肌球蛋白X干涉载体的构建与鉴定

肌球蛋白X(Myosin X,Myo X)是近年来发现的在细胞运动中发挥重要功能的非传统型肌球蛋白,但其发挥功能的分子机制目前尚不明确,RNA干扰技术是目前研究基因功能常用的方法。构建针对鼠的Myo X基因mRNA的shRNA真核表达载体,并转染NLT细胞,观察其对Myo X表达的抑制效应。结果显示,shRNA-2能够有效地降低Myo X的蛋白表达水平,而shRNA-1则不能。进一步分析显示,转染shRNA-2的NLT细胞丝状伪足的形成受到抑制,表明shRNA-2是能够抑制Myo X表达的干涉载体,此干涉载体的构建为进一步研究Myo X的新功能,揭示其作用的分子机制奠定基础。     

刺参管足SMART cDNA文库构建及EST分析

以大连广鹿岛野生刺参的管足mRNA为材料,利用SMART cDNA Construction Kit构建了表达型cDNA文库.初始文库的滴度为1.3 × 106PFU/mL,蓝白斑估测量组率合格.扩增后获得96 mL文库,滴度为1.8 × 1010PFU/mL,从扩增文库中随机挑取200个噬菌斑进行PCR检测,电泳检测结果表明重组率大于90％,片段大小在0.25 -2.0 kb之间的插入片段占80.4％,文库构建成功.随机选择122个噬菌斑转化为质拉pTriplEx2并测序,测序成功率83.6％,软件处理后100 bp以上的clean ESTs有72条,测通的为65条,占成功测序样品的63.7％.72条序列经SeqMan软件拼接,获得48条conting/singletons,在线Blast比对发现其中26条具有同源序列并获得注释,另外20条可能为新基因,其功能和结构有待进一步利用生物信息学的方法进行分析和研究.     

绿色糖单孢菌木质素过氧化物酶的分离纯化及鉴定

木质素过氧化物酶是一种重要的具有工业应用前景的木质素降解酶,但已报道真菌来源的木质素过氧化物酶只能在酸性低温条件下发挥作用,限制了其进一步的工业应用.通过培养一株耐热耐碱放线茵——绿色糖单孢茵发酵产酶,采用DEAE-Cellulose,CM-Cellulose和Superdex 75凝胶过滤层析等分离纯化方法,得到一种具有耐热耐碱特性的木质素过氧化物酶.经凝胶电泳检测其为单一蛋白,分子量为41 kD.最终纯化倍数达到20倍,活性回收率为6％.采用LTQ法对纯酶进行蛋白质归类鉴定,得到其部分氨基酸片段,为该酶的进一步分子生物学研究奠定基础.