应用抑制消减杂交分离雌、雄鸡胚性分化早期的差异表达基因

分离不同性别的鸡胚性分化早期差异表达基因,可为禽类性别决定和性分化机制研究提供基本信息。本研究分别以孵化3.5-6d的雌、雄鸡胚性腺为材料,利用抑制性消减杂交技术成功构建了雌-雄鸡胚间正、反向消减cDNA文库,并利用斑点印迹杂交从中筛选出了39个性别差异表达的阳性cDNA克隆。以持家基因GAPDH为参照指标检测消减文库的消减效率,结果发现两个文库的消减效率均高达25倍。插入片段PCR鉴定结果显示,消减文库中cDNA插入片段的长度主要分布于250-750bp之间。分别对雌、雄鸡胚消减文库中的252和168个cDNA克隆进行斑点杂交筛选,再随机从两个消减文库中共抽取39个阳性差异表达克隆进行序列测定及序列比对分析。结果表明:这39个cDNA克隆分别代表了定位于鸡不同染色体上的18个已知功能的基因和11个假定基因;参照哺乳动物同源基因的功能,所得的18个已知差异基因可能参与多种生物反应过程。用半定量RT-PCR方法对雌、雄鸡胚消减文库中各5个基因的表达情况进行进一步验证,发现除雌性库中的一个基因外其它9个基因均有较明显的性别差异表达。这些性别差异表达基因的获得为进一步研究鸡胚性腺发育中的基因表达调控奠定了基础     

芜菁消减文库特异基因片段功能的初步分析

目的：对4个消减cDNA文库中筛选到的特异基因片段进行功能分析,为筛选津田芜菁和赤丸芜菁花青素合成的特异基因和代谢途径奠定基础。方法：以不同处理的津田芜菁和赤丸芜菁块根为材料,采用抑制削减杂交构建4个消减cDNA文库并富集特异基因群体,同时对消减文库的特异基因片段进行初步的生物信息学分析。结果：通过功能聚类、代谢途径分析和基因注释等手段分析了消减cDNA文库的特异基因片段。结论：对消减文库特异基因片段的功能分析,为进一步分离和鉴定依光型和非依光型花青素合成相关基因奠定了基础。     

肾癌相关基因克隆——肾癌cDNA消减文库的构建

应用抑制性消减杂交技术,构建人肾癌与正常肾差异表达的cDNA消减文库.分别从肾癌及正常肾细胞系中提取poly(A)⁺RNA,依次合成单链及双链cDNA,经酶切成平均大小为400～600 bp的片段,将肾癌cDNA分为两组,分别与两种不同的接头衔接,再与正常肾cDNA进行两次消减杂交及两次抑制性PCR后,将产物与T/A载体连接构建成功cDNA消减文库,并转染大肠杆菌进行文库扩增.构建成功具有高消减效率的人肾癌cDNA消减文库,非特异性cDNA片段被有效地消减,特异表达的cDNA得到富集.文库扩增后得到6 500个克隆,随机挑取350个制备质粒,酶切分析均得到400～600 bp插入片段.所构建的人肾癌cDNA消减文库为进一步大批量筛选、克隆肾癌特异性表达的未知新基因奠定了基础.     

快速老化模型小鼠海马正反向抑制消减cDNA文库的构建

目的:构建快速老化模型小鼠(SAM)海马正反向抑制消减cDNA文库,以揭示SAMP8学习记忆脑老化的机制,同时为研究阿尔茨海默病(AD)的发病机制提供线索。方法:以快速老化模型小鼠SAMP8和SAMR1海马的总RNA为材料,采用抑制消减杂交方法和蓝白斑筛选克隆构建文库,并用PCR鉴定了文库的质量。结果:成功构建了12月龄雄性SAMP8和SAMR1海马的正反向抑制消减cDNA文库,其中正向文库包含864个克隆,反向文库包含960个克隆,阳性克隆率为96.16%,插入片段范围为250～2000bp。结论:SAMP8和SAMR1海马的正反向抑制消减cDNA文库的构建,为进一步筛选鉴定SAMR1和SAMP8海马差异表达基因提供了丰富的实验材料。     

SSH 法结合定量 PCR 技术研究双肌臀猪 肌肉组织的差异表达基因

利用抑制性消减杂交技术 (suppression subtractive hybridization , SSH) 成功地构建了双肌臀与非双肌臀大白猪肌肉组织差异表达的消减 cDNA 文库,获得有效克隆 686 个. 对整个文库测序分析,共获得 587 条有效序列. 利用 BLAST 在线软件与 GenBank、 GenBank EST 和 Tigr Porcine EST 等数据库进行同源序列比较,发现其中有 11 个未知新序列,可能代表“双肌臀”大白猪肌肉组织特异表达的新基因. 对文库中所包含的兰尼啶受体基因 (RYR1)、钙依赖性蛋白激酶 基因 (CAMK2)、人类胰岛素生长因子结合蛋白 -7 基因 (IGFBP7),以及 695号、 882号和480号3个新基因进行了实时荧光定量 PCR 检测,结果显示： RYR1、 CAMK2 及 IGFBP7 基因在双肌臀猪肌肉组织 RNA 池中的表达量,分别为对照的非双肌臀猪肌肉组织 RNA 池中表达量的 1.87、 1.90 和 1.85 倍; 695 号、 882 号和 480 号 3 个新的 ESTs 在双肌臀猪肌肉组织 RNA 池中的表达量为非双肌臀猪肌肉组织 RNA 池中表达量的 1.48、 1.44 和 1.78 倍. 这提示我们,上述基因的上调表达很可能与猪双肌臀性状的发生有密切的关系,可以作为研究该性状的候选基因.     

用改良消减杂交结合选择性PCR法构建老年性白内障消减cDNA文库

为构建含较多大片段的高质量的老年性白内障消减cDNA文库 ,利用生物素标记、磁珠分离的改良消减杂交法获得差异cDNA .利用选择性PCR法扩增其中大片段差异cDNA ,将其与T 载体进行T A连接并转化入大肠杆菌 ,成功构建老年性白内障消减cDNA文库 .共获得 4 0 0 0余个克隆 ,随机挑取的 2 2个克隆中 ,≥ 10 0 0bp的片段有 7个 ,占 31 8% ,≥ 75 0bp有 15个 ,占 6 8 2 % .将≥ 75 0bp的 15个克隆进行反向点杂交 ,排除其中假阳性克隆 ,阳性克隆经测序并与GenBank比较 ,得到 6个已知基因、1个新基因 ,6个已知基因中 4个为全长基因 ,说明所得cDNA片段较大 ,文库质量较高 .改良消减杂交法结合选择性PCR法可以快速有效地获得大片段高质量的消减cDNA文库 ,为进一步筛选、鉴定老年性白内障致病相关基因奠定了基础     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

应用抑制性消减杂交技术筛选流感病毒感染宿主应答基因

从宿主系统寻找病毒感染特异性相关的生物大分子是研究病毒药物靶标和诊断标志物的新方向 .为了筛选宿主细胞中流感病毒感染特异性基因 ,采用抑制性消减杂交技术 (SSH) ,以流感病毒A 鲁防 93 9(H3N2 )感染MDCK细胞及正常MDCK细胞为材料 ,构建病毒感染特异性差减cDNA文库 ,PCR法扩增鉴定其中插入片段大小 .从差减文库中随机挑取 10 0个克隆进行测序 ,用生物信息学方法对其同源性和基因功能进行分析和预测 .结果显示 ,成功构建了流感病毒感染特异性差减cDNA文库 ,文库中cDNA片段长度在 2 5 0～ 10 0 0bp之间 .从文库中随机选取 10 0个克隆测序 ,获得了 95个有效序列 ,经blast同源性分析发现 ,大部分基因为参与宿主细胞能量代谢和蛋白质生物合成过程中的基因 ;其中 19个为无任何功能线索的新基因片段 .流感病毒感染特异性差减cDNA文库的建立和筛选出病毒感染应答候选新基因cDNA片段 ,为发现新型流感病毒药靶和诊断标志物以及病毒感染机制研究打下基础     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫（Eimeria tenella）孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交（SSH）技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示：从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。     

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、...     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭未草地放牧演替的生理机制有重要意义,大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段,放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势,牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关,放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关。     

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     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

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。     

Morphology and putative function of the colon and cloaca of marine and freshwater snakes

Among tetrapods, evidence for postrenal modification of the urine by the distal digestive tract (including the colon and cloaca) is highly variable. Birds and bladderless reptiles are of interest because the colon and cloaca represent the only sites from which water and ions can be reclaimed from the urine secreted by the kidney. For animals occupying desiccating environments (e.g., deserts and marine environments), postrenal modification of the urine may directly contribute to the maintenance of hypo‐osmotic body fluids. We compared the morphology and distribution of key proteins in the colon, cloaca, and urogenital ducts of watersnakes from marine (Nerodia clarkii clarkii) and freshwater (Nerodia fasciata) habitats. Specifically, we examined the epithelia of each tissue for evidence of mucus production by examining the distribution of mucopolysaccharides, and for evidence of water/ion regulation by examining the distribution of Na⁺/K⁺‐ATPase (NKA), Na⁺/K⁺/Cl^? cotransporter (NKCC), and aquaporin 3 (AQP3). NKCC localized to the basolateral epithelium of the colon, urodeal sphincter, and proctodeum, consistent with a role in secretion of Na⁺, Cl^?, and K⁺ from the tissue, but NKA was not detected in the colon or any compartment of the cloaca. Interestingly, NKA was detected in the basolateral epithelium of the ureters, suggesting the urothelium may play a role in active ion transport. AQP3 was detected in the ureters and coprodeal complex, consistent with a role in urinary and fecal dehydration or, potentially, in the production of the watery component of the mucus secreted by the coprodeal complex. Since no differences in general cloacal morphology, production of mucus, or the distribution of ion transporters/water channels were detected between the two species, cloacal osmoregulation may either be regulated by proteins not examined in this study or may not be responsible for the differential success of N. c. clarkii and N. fasciata in marine habitats. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.