乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒的构建及鉴定

目的：构建乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。方法：乙酰肝素酶cDNA全长扩增和最佳siRNA干扰片段筛选分别采用PCR和Real-time PCR方法,慢病毒系统载体分别使用pWPI和siRNA pSico系统,采用限制性内切酶快速连接方法联接目的基因和最佳最佳siRNA干扰片段,表达载体鉴定均采用核苷酸序列测定,HPSE重组慢病毒表达质粒和siRNA片段细胞转染采用脂质体转染法。结果：成功扩增乙酰肝素酶全长并连接入pWPI载体构建成重组表达载体HPSE-pWPI,重组质粒测序结果显与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。筛选出最佳siRNA干扰片段为HPSE-1222并成功插入pSico载体,构建成重组表达载体HPSE-siRNA pSico,重组载体测序显示与构建的shRNA结构序列完全一致。结论：成功采用慢病毒载体系统构建了乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Identity of an ABA-activated 46 kDa mitogen-activated protein kinase from <Emphasis Type="Italic">Zea mays</Emphasis> leaves: partial purification,identification and characterization

Mitogen-activated protein kinase (MAPK) cascades have been shown to be important components in abscisic acid (ABA) signal transduction pathway. In this study, a 46 kDa MAPK (p46MAPK) induced by ABA was partially purified from maize (Zea mays) by Q-Sepharose FF, Phenyl-Sepharose FF, Resource Q, Mono QTM 5/50 GL, poly-l-lysine-agarose, and Superdex 75 prep-grade columns, and was identified as ZmMAPK5 (gi|4239889) by the matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. Furthermore, the kinase showed optimal activity at pH 8.0, 30°C, and 10 mM MgCl₂; the K _m for myelin basic protein (MBP) substrate and ATP were 0.13 μg μl⁻¹ and 62 μM, respectively. MBP was the preferred substrate, of which the threonine residue was phosphorylated. Finally, the kinase was found to respond to diverse extracelluar stimuli. These results enable us to further reveal the function of the ZmMAPK5 in ABA signaling. The authors Haidong Ding and Aying Zhang contributed equally to the work.     

焕镛木小孢子发生及雄配子体发育研究

运用常规石蜡切片法和细胞学压片法,对焕镛木[Woonyoungia septentrionalis (Dandy) Law]小孢子发生、雄配子体发育以及花粉萌发进行观察分析.结果显示:焕镛木花单性,雄花的雄蕊多数、离生,每一花药具4个花粉囊,花药壁5～7层.腺质绒毡层具1～2层细胞;小孢子形成时胞质分裂方式为修饰性同时型;四分体排列方式以四面体型为主,并有交叉型和左右对称型,偶见T型和直线型;成熟花粉粒为二细胞型花粉粒.小孢子发生和雄配子体发育过程中存在变形及败育现象.花粉萌发率为79.89%～80.62%.结合焕镛木花粉粒数目和萌发率的分析认为,雄配子体发育中的败育不是影响焕镛木濒危的主要因素.     

川西亚高山红桦-岷江冷杉林优势种群的空间格局分析

以红桦-岷江冷杉林4 hm²样地调查数据为基础,采用点格局分析法O-ring函数,分析了红桦和岷江冷杉2个优势种群不同龄级的空间分布格局,以及各龄级间的种内和种间关联性.结果表明：红桦种群径级分布呈单峰型,为衰退种群;岷江冷杉种群径级分布呈倒“J”型,为进展种群.岷江冷杉与红桦大树在所有尺度上均为随机分布,其余龄级在小尺度上为聚集分布,随尺度增大趋于随机分布和均匀分布,最大聚集强度随龄级增大而减弱.空间关联主要发生在小尺度上.岷江冷杉种内关联以小尺度正关联为主,红桦种内关联以小尺度负关联为主.对于2个种群的相同龄级,小龄级、中龄级和大龄级间,分别以空间无关联、负关联和无关联为主.2个种群间不同龄级间以负关联为主,龄级相差越大,其负关联越强.     

利用黑色斑蚕作亲本、选育日系普斑限性品系

根据家蚕Bombyx moli斑纹互作原理,利用2032限性品种的雌,与自然突变体雄杂交。F2代出现分离,于是淘汰所有黑色斑蚕,只留下普斑蚕和素斑蚕,即得到新限性普斑系。新限性品系得到后,做连续3代的系统选育,其中F3代为蛾区混合育,F4～F5代采用单蛾育。蛾区混合育着重个体选择,单蛾育以蛾区选择为主,个体选择为辅。性状基本稳定后,即初步对其作主要经济性状的测定。结果显示:新限性品系在5龄经过、全龄经过上比两亲本略短。在全茧量、茧层量、茧层率几项指标上较两亲本为优,分别比两亲本平均值提高31%、38%、5%。     

大气二氧化碳倍增对短葶飞蓬生长和有效成分积累的影响

通过对人工倍增CO₂浓度（800±100） μmol·mol^-1与正常CO₂浓度（400±25） μmol·mol^-1条件下短葶飞蓬植株生物量、体内灯盏乙素和总咖啡酸酯含量及产量的比较,探讨了CO₂浓度升高对药用植物短葶飞蓬生物量、有效成分含量及产量的影响.结果表明：CO₂浓度倍增条件下,短葶飞蓬植株生物量增加了22%;总咖啡酸酯及灯盏乙素含量分别增加23%和26%,产量分别增加37.6%和45.3%.不同器官的生物量及有效成分含量对CO₂浓度升高的响应不同.在CO₂浓度倍增条件下,短葶飞蓬植株含氮量减少47.2%,与次生代谢有效成分含量的变化呈反比,符合“碳素/营养平衡假说”;植株生物量与次生代谢产物含量的变化呈正相关,植株生长与次生代谢产物积累之间没有表现出权衡关系.说明合理施用CO₂是可实现短葶飞蓬的优质高产.     

模拟酸雨胁迫对青冈幼苗光合特性和叶绿素荧光参数的影响

选择亚热带常绿阔叶林优势树种青冈幼苗为研究对象,设置3个酸雨处理：pH 2.5、pH 4.0、pH 5.6(CK),研究不同强度的模拟酸雨对青冈幼苗光合特性、叶绿素荧光参数和叶绿素含量的影响.结果表明：经过2年酸雨处理,青冈的净光合速率随着酸雨强度的增加而显著上升.pH 2.5、pH 4.0处理的酸雨增大了青冈的气孔导度和蒸腾速率,且对pH 2.5处理的影响更为显著.胞间CO₂浓度的大小顺序是pH 2.5>pH 5.6>pH 4.0.pH 2.5、pH 4.0处理青冈的最大净光合速率、光补偿点、光饱和点、暗呼吸速率显著高于对照,表观量子效率对酸雨胁迫不敏感.pH 2.5、pH 4.0处理青冈的PSⅡ原初光能转化效率和PSⅡ的潜在活性显著高于对照.青冈的叶绿素相对含量大小顺序是pH 2.5>pH 5.6>pH 4.0,且pH 2.5与pH 4.0之间有显著差异.说明青冈幼苗的光合参数、叶绿素荧光参数指标在pH 2.5和pH 4.0的酸雨处理下有所增加,且在pH 2.5强度下增加更为明显.