猪链球菌2型菌毛骨架蛋白编码基因SSU2101敲除突变株的构建及其生物学功能

利用同源重组基因敲除方法构建猪链球菌2型(Streptococcus suis serotype2,S.suis2)中国强毒株05ZYH33菌毛骨架蛋白(Backbone protein,BP)编码基因SSU2101敲除突变株。采用引物特异性PCR分析、Southern杂交及RT-PCR等方法鉴定,证实成功构建了BP基因缺失突变株。生物学特性显示,突变株的菌落形态、溶血活性以及染色特性方面与野生株之间均无明显差异。小鼠致病性试验结果显示,突变株的毒力比野生株显著减弱。研究结果提示菌毛在S.suis2感染致病过程中起重要作用,为系统研究S.suis2菌毛分子装配机制及其生物学功能奠定了基础。     

The formation of improved tetraploid population of red crucian carp × common carp hybrids by androgenesis

Bisexual fertile diploid androgenetic individuals (A0) (2n=100) were formed by androgenesis. In this way, the diploid spermatozoa from male allotetraploid hybrids (AT) (4n=200) of red crucian carp (Carassius auratus red var.) (♀) × common carp (Cyprinus carpio L.) (♂) were used to fertilize the UV-treated hap- loid eggs of goldfish (Carassius auratus), and living androgenetic diploid fish were developed. The A0 became sexually mature at the age of 2 years, and they fertilized with each other to form their offspring (A1). In this study, we observed the chromosomal number, gonadal structure and appearance of A1 fish. The results are as follows: (1) In A1, there were 85% tetraploids (A1-4n), 10% triploids (A1-3n) and 5% diploids (A1-2n), suggesting that diploid A0 could produce diploid gametes. It was concluded that the formation of diploid gametes generated from diploid A0 was probably related to the mechanism of pre-meiotic endoreduplication. (2) Among A1, only A1-4n possessed normal ovaries and testes. The mature males of A1-4n produced white semen. Under the electron microscope, the head of diploid sperm generated by A1-4n was bigger than that of haploid sperm generated by red crucian carp. In the testes of the A1-4n, there were many mature normal spermatozoa with a head bearing plasma mem- brane and a tail having the typical structure of "9 2" microtubules. Between the head and the tail, there were some mitochondria. The ovaries of A1-4n developed well and mainly contained II, III and IV-stage oocytes. The IV-stage oocytes were surrounded by inner and outer follicular cells. The micropyle was observed on the oolemma of follicular cells. There were abundant yolks and plenty of endoplasmic reticulum in the cytoplasm of IV-stage oocytes. Because A1-2n and A1-3n were distant crossing diploid hybrids and triploid hybrids respectively, they possessed abnormal gonads, and no mature semen and eggs were observed. (3) Compared with allotetraploids, the A1-4n fish not only had advantages such as fast growth rate and strong resistibility but also showed some new good performances such as high ratio of body width to body length, smaller heads and shorter tails. These results indicated that an- drogenesis could produce bisexual fertile tetraploids and improve the shape of allotetraploid hybrids as well, which will be of great significance in both the cell genetics research and fish breeding.     

高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca2+-ATPase、Mg2+-ATPase及Ca2+分布的影响

将水培后盆栽的花生幼苗,置于培养箱42℃高温培养,定时测定幼苗叶光合速率、叶绿素含量和叶绿体Ca2 -ATPase、Mg2 -ATPase的相对活性,并观察幼叶细胞内Ca2 分布的变化。试验结果表明:高温胁迫过程中,光合速率及叶绿素含量都随处理时间的延伸而下降,并呈显著正相关;叶绿体Ca2 -ATPase和Mg2 -ATPase高温胁迫过程中相对活性呈先升后降趋势,Ca2 -ATPase热敏性高于Mg2 -ATPase;高温胁迫过程中,Ca2 具有从胞外转运到胞质内和叶绿体中的趋势,Ca2 能够稳定高温胁迫下叶肉细胞膜和叶绿体的超微结构。     

稻鸭共作有机农业模式的能值评估

应用能值分析方法对稻鸭共作有机农业模式（模式Ⅰ）和对应的稻麦常规生产模式（模式Ⅱ）进行比较研究,并比较了两种模式的生态经济效益.结果表明,模式Ⅰ的能值效益高,自组织能力、可持续发展能力强,产品安全性高,净能值产出率（EYR）、系统产出能值反馈率（FYE）、能值可持续指标（ESI）分别是模式Ⅱ的1.57、14.1和8.71倍;基于能值的产品安全性指标（EIPS）模式Ⅰ为0,模式Ⅱ为-0.66;模式Ⅰ对环境的压力小于模式Ⅱ,能值投资率（EIR）,环境负载率（ELR）分别是模式Ⅱ的40.1%和18.3%;但模式Ⅰ的经济效益低于模式Ⅱ,其产出、毛收入和净效益分别低于模式Ⅱ15.7%、9.6%和29.6%;以能值 货币价值计算,模式Ⅰ的产投比、毛收入和净收入分别高于模式Ⅱ50%、102.6%和136.4%.随着生产系统的优化和市场对有机食品认知程度的提高,模式Ⅰ的经济效益具有提高的潜力.     

遮荫对掌叶半夏生长、产量及光合生理特性的影响研究

目的:研究遮荫对掌叶半夏生长、药材产量和光合生理指标的影响,分析适宜掌叶半夏药材高产的遮荫度,探寻其产量差异形成的光合生理机制。方法:采用遮荫网遮荫,共设置了5种光强处理,透光率分别为全光对照CK(100%)、T1(75%)、T2(65%)、T3(50%)、T4(25%),测定各遮荫处理下掌叶半夏的株高、单株叶片数、单株叶面积、单片叶面积、药材产量、叶绿素含量、光合日进程和叶绿素荧光参数。结果:(1)遮荫可显著(P<0.01)提高掌叶半夏的株高、单株叶面积和叶绿素含量;(2)光合日进程曲线测定结果表明,除T4处理外其他处理均发生光合"午休"现象,日平均光合速率(Pn)以T1处理最高,绝对光能利用率(Eu)以T3处理最高;(3)叶绿素荧光参数测定结果表明,CK的Fv/Fm最低,发生的原因可能与PSⅡ反应中心的光化学伤害有关;(4)药材产量以T2处理最高,较CK增产56.34%。结论:掌叶半夏更适宜在遮荫条件下栽培,透光率在65%时有利于掌叶半夏药材高产,其产量差异的形成可能与日均光合速率的不均衡有关。     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

光强对浒苔充气叶状体内氧气浓度的影响

以烟台海域定生浒苔为研究对象,采用光暗转换法,研究了不同光强条件下浒苔充气叶状体内部氧气浓度的变化。结果表明,浒苔充气叶状体内部气体成分中含有氧气,其浓度在360μmol/m2.s光强下达到最高值且保持稳定,光暗转换法显示其最高稳态到最低稳态的过渡仅需约10 s,此外,最低稳态的氧浓度仍高于水体饱和溶氧浓度。     

一种不依赖钙离子的酸性α淀粉酶基因的克隆表达

以产酸性淀粉酶菌株Bacillus sp.CN7的基因组DNA为模板,PCR扩增到α淀粉酶成熟肽基因,将该基因插入表达质粒pSE380中,构建重组质粒pSE380-cn7a。将重组质粒导入到Escherichia coli JM109中,IPTG诱导表达。重组酶经Sephacryl S300、Ni-NTA纯化后测定其酶学性质。重组酶CN7A的最适温度为65°C,最适pH为5.5?6.0,对可溶性淀粉的Km值为3.784g/L,最大反应速度为101.2mg/(L·min),该酶的热稳定性不依赖钙离子。     

血浆脑钠肽对不同年龄急性冠脉综合征患者介入治疗术后临床预后的意义

目的：探讨并比较血浆脑钠肽（BNP）对不同年龄组急性冠脉综合征（ACS）患者经皮冠脉介入（PCI）术后临床主要不良心脏事件（MACE）的预测价值。方法：回顾分析2007年12月至2010年3月因ACS在我院行PCI的患者,分为260岁和〈60岁两组,入院常规检验BNP,随访住院期间及出院后6个月内MACE（包括死亡、心源性休克、再发心绞痛、非致命性再次心肌梗死、非致命性心力衰竭、心律失常、靶血管血运重建、再次入院复合终点）的发生情况。结果：随访269例260岁患者,失访7例（2．6％）,118例〈60岁患者,失访3例（2．5％）,262例≥60岁患者的年龄与BNP水平显著正相关（r=0．368,P〈0．01）,而115例〈60岁患者无显著相关性（r=-0．014,P〉0．05）。年龄≥60岁患者BNP水平与〈60岁患者比较,差异显著（P〈0．01）,年龄260岁患者MACE与〈60岁患者比较,差异显著（P〈0．01）,两组患者BNP水平与LVEF呈显著负相关（P均〈0．01）。多因素logistic回归分析显示：BNP可独立预测两组患者住院期间及出院后6个月内MACE（≥60岁OR=4．369P〈0．01;〈60岁OR=7．773P〈0．01）发生率。两组患者BNP水平对MACE预测的ROC曲线结果比较,差异无统计学意义（P〉0．05）。结论：不同年龄患者BNP水平及MACE发生率存在差异,但年龄并不影响BNP对ACS患者临床预后的预测价值。     

14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.