斑鳜myostatin基因及其启动子的克隆与序列分析

采用Tail-PCR和常规PCR技术首次克隆出斑鳜myostatin基因及其启动子序列。经生物信息学分析发现,斑鳜myostatin基因由3个外显子和2个内含子组成,编码区1131bp,共编码376个氨基酸。斑鳜myostatin启动子区域大小为840bp,存在1个TATAA-box、4个E-box和1个CAAT-box作用元件。利用软件CLUSTALW和MEGA3.1构建14种硬骨鱼的myostatin启动子的系统进化树结果表明:斑鳜同大口黑鲈亲缘关系最近,与鲤鱼和缨野鲮亲缘关系较远,其结果与传统形态学分类中的亲缘关系一致。斑鳜myostatin基因及其启动子克隆与特征分析,将为进一步研究鱼类myostatin基因的表达调控及其功能分析提供参考。     

中华根瘤菌NP1氨单加氧酶基因的克隆与功能鉴定

以中华根瘤菌NP1(Sinorhizobium sp.NP1)为原始菌株,通过同源克隆与Tail-PCR方法,获得1089bp的氨单加氧酶基因(amo)全长序列.该基因编码362个氨基酸,其二级结构与Sinorhizobium meliloti1021AMO的二级结构相似,该蛋白有9个跨膜区段.以自杀穿梭质粒pJQ200SK为原始载体,构建NP1amo基因敲除质粒pJQ200SK-amo-Tc.采用三亲本杂交的方法将该质粒转入原始菌株NP1中,获得amo基因敲除菌株NP1∷amo.通过本贝洛氏(Berthelot)法对氨氮进行测定,发现NP1∷amo的脱氮效率比原始菌株NP1下降约35%.该结果表明,本实验中所克隆的氨单加氧酶基因为脱氮关键酶基因.     

杜氏盐藻泛素基因cDNA的克隆及系统进化分析

根据玉米,水稻等物种泛素序列设计一对简并引物.提取杜氏盐藻细胞的总RNA,利用RT-PCR方法扩增盐藻泛素基因的cDNA片断,回收两个长度不同的片断ubi-1和ubi-2,将其克隆到pMDl8-T载体上,测序后进行序列分析,为克隆杜氏盐藻泛素基因的cDNA序列并进行进化分析.结果 ubi-1经测序后得到一个完整拷贝(228 bp)和一个不完整的泛素cDNA序列(191 bp).Ubi-2经测序后得到两个拷贝(556 bp)和一个不完整的泛素cDNA序列(191 bp).盐藻3个不同拷贝泛素cDNA序列之间存在差异,但所编码氨基酸序列相同.盐藻泛素cDNA序列与其他物种的泛素cDNA序列具有高的同源(70%～85%),所推导的氨基酸序列与其他物种仅存在1～2个氨基酸的差异.进化分析显示,所分离的盐藻泛素基因与两个模式生物果蝇和衣藻的泛素基因共处一个进化支,彼此亲缘关系最近.盐藻泛素基因与其他物种的泛素基因可能来自共同的"祖先"基因.在进化中高度保守.     

小麦CPP转录因子基因的电子克隆及生物信息学分析

目的:运用电子克隆的方法获得小麦中的CPP转录因子基因。方法:以水稻的CPP转录因子作为探针,对小麦的EST数据库进行搜索,应用相关软件进行聚类分析、拼接组装和延长。结果:克隆出4个小麦CPP基因,分别命名为TaCPP1、TaCPP2、TaCPP3和TaCPP4,序列长度分别为977bp、3 022bp、1 582bp和1 156bp,开放阅读框为975bp、2 298bp、720bp和750bp。4个CPP类型蛋白质都具有一个或两个CXC域,而且多数还拥有完整的CRC结构域。结论:4个小麦CPP基因与水稻CPP蛋白具有高度的同源性。研究结果为进一步实验克隆和研究其功能提供了理论基础。     

牛Irak-1基因的电子克隆及生物信息学分析

电子克隆提供了一种利用基因组数据库克隆新基因全长cDNA序列的策略。利用小鼠Irak-1基因编码序列(NM_008363)为种子序列进行电子克隆获得了牛Irak-1基因完整编码序列。然后,用生物信息学方法分析了该基因的结构,微卫星位点,密码子偏性和氨基酸的同源性等。结果表明:该基因cDNA全长2 645bp,无内含子,最大开放阅读框2 157bp,编码718个氨基酸,与小鼠的同源性为77%。     

小菜蛾线粒体复合物Ⅲ铁硫蛋白基因5'上游调控区域的克隆及序列分析

利用Tail-PCR技术克隆了小菜蛾线粒体复合物Ⅲ铁硫蛋白亚基的基因组DNA序列,全长5 013 bp,GenBank 登录号为HM210791.研究表明,该基因具有3个内含子,大小分别为96 bp、577 bp和549 bp,且其内含子两端具有典型的GT-AG结构.生物信息学分析表明,该基因5'上游调控区域不仅包括TATA-box等启动子核心序列,而且含有BR-C Z4、Hb、Dfd、CF2-Ⅱ和HSF等转录因子结合位点.进一步鉴定了该基因的转录起始位点、启动子序列和CPG岛区域.为小菜蛾线粒体复合物Ⅲ铁硫蛋白亚基的转录调控机制和具体的生理功能研究提供一定基础.     

莲藕CBF2基因cDNA克隆及序列分析

目的:克隆莲藕CBF2基因的c DNA全长并对其进行序列分析。方法:根据已有的ESTs序列,设计3'和5'端RACE引物,运用RACE技术克隆莲藕CBF2基因c DNA全长。结果:克隆得到全长为1 560bp c DNA序列,其中包括350bp的3'非编码区和124bp的5'非编码区及一个1 086 bp的完整开放阅读框,编码362个氨基酸,序列末端有poly(A)尾。其核苷酸序列与NCBI数据库中大豆DREB2C/CBF2、马铃薯DREB2A/CBF2、黄瓜DREB2A/CBF2及花生DREB2A/CBF2的同源性较高,分别为83%、77%、76%和76%,因此把该基因命名为Lr CBF2。氨基酸序列进化树分析表明该基因与葡萄相关基因亲缘关系较近。结论:分离克隆得到莲藕CBF2基因,为进一步研究莲藕中该基因的功能奠定基础。     

人天然免疫基因BCL10的猪同源物的识别、克隆与初步表达分析

采用电子克隆方法克隆到大小为925 bp的人天然免疫蛋白BCL10的猪同源基因完整cDNA序列(GenBank登录号：EU088132), 并利用RT-PCR方法从猪的全血中扩增出包含702 bp的完整开放读码框架(ORF)的cDNA片段。经核酸测序, 证明与电子克隆结果相符。利用NCBI BLAST分析该cDNA包含3个大小为57 bp、289 bp和356 bp的外显子, 并且定位于猪的4号染色体上。采用半定量PCR技术检测基础水平猪各组织BCL10基因mRNA表达丰度, 并将该基因构建到带有绿色标签的真核表达载体pEGFP-C1中, 采用脂质体转染法将该基因转入PK-15细胞, 通过绿色荧光标记和RT-PCR方法检测实验组的BCL10蛋白表达。研究结果表明, BCL10基因mRNA在脾脏中表达最高; 胸腺、大脑和淋巴结表达次之, 而肝脏只有微量表达, 肾脏没有检测到表达; 同时BCL10基因在PK-15细胞中得到了有效表达。     

甘蔗ATP合酶基因的电子克隆及生物信息学分析

目的:运用电子克隆的方法获得甘蔗中的ATP合酶基因。方法:以小麦中一个ATP合酶基因为种子序列,对甘蔗的EST数据库进行搜索,应用相关软件进行聚类分析、拼接组装和延长。结果:获得一个ATP合酶基因SATPC1的cDNA序列全长,该序列长1 415bp,包含一个完整的1 077bp的ORF,编码358个氨基酸,且与水稻、玉米、高粱和葡萄等其他植物的ATP合酶具有高度的同源性。结论:电子克隆获得的cDNA序列为完整的甘蔗ATP合酶基因全长cDNA。     

牛Pbx-1基因的电子克隆及生物信息学分析

新基因全长cDNA序列很难获得,但电子克隆却提供了基因克隆的一种策略.利用小鼠Pbx-1基因编码序列(NM_183355)为种子序列进行电子克隆获得牛Pbx-1基因完整编码序列.然后,用生物信息学方法分析了牛的Pbx-1基因的结构,密码子偏性和氨基酸的同源性等.结果表明:该基因cDNA全长1 754 bp,无内含子,最大开放阅读框1 305 bp.编码434个氨基酸.预测其编码的蛋白分子量为47 189.5 Da,与小鼠的同源性为81%.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000