Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleurotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBlue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P. nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a ＂reporter＂ gene, green fluorescent protein gene can be successfully stably exoressed in this Pleurotus species.     

产丁醇大肠杆菌工程菌的构建

【目的】为了在大肠杆菌(Escherichia coli)中导入改良的丁醇合成途径,使非生产菌株大肠杆菌具备产丁醇的能力。【方法】克隆大肠杆菌乙酰转移酶基因atoB和丙酮丁醇梭菌(Clostridium acetobutylicum)丁醇合成途径关键酶基因(crt、hbd、adhE),构建多顺反子表达质粒pSE380-atoB-adhE-crt-hbd;克隆齿垢密螺旋体(Treponema denticola)反式烯酰辅酶A还原酶基因ter,构建表达质粒pSTV29-ter,并将双质粒导入到大肠杆菌。【结果】构建的工程菌能半厌氧发酵产微量丁醇,产量为0.08g/L。【结论】大肠杆菌中的丁醇合成途径导入成功,构建了产丁醇的大肠杆菌工程菌。     

Green fluorescent protein （GFP） transsenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm animals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfecUon system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe- tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h resulted in the highest transfecUon rate （3.6%）. The percentage of GFP reconstructed embryos that de- veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Reconstructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 delivered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.     

人MiTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MiTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MiTERF3基因5''侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体pGL6-TA,构建人MiTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与GenBank报道的一致,且插入方向正确。含人MiTERF3基因启动子的报告基因荧光素酶的表达活性显著提高（P<0.05）,约为对照组（空载体pGL6-TA）的9.8倍。本研究通过对人MiTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MiTERF3基因表达的调控机制奠定实验基础。     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Scriptaid affects histone acetylation and the expression of development-related genes at different stages of porcine somatic cell nuclear transfer embryo during early development

Although the somatic cell nuclear transfer(SCNT) technique has been used extensively for cloning and generating transgenic pigs,the cloning efficiency is still very low.It has been proposed that the low efficiency of this technique is the result of incomplete epigenetic reprogramming and abnormal gene expression during early embryonic development.In this study,we investigate the effect of Scriptaid,a low-toxicity histone deacetylase inhibitor,on the developmental competence of porcine SCNT embryos.We found that treating SCNT embryos with 500 nmol/L Scriptaid for 15 h after activation significantly enhanced the blastocyst formation rate(27.7%) compared with the untreated group(control)(12.2%,P<0.05).Using an immunofluorescence technique to measure the average fluorescence intensity,we also found that treating SCNT embryos with Scriptaid increased the level of histone acetylation on histone H3 at lysine 14(acH3K14).Furthermore,treating embryos with Scriptaid increased the expression level of three genes that play important roles during embryonic development(Oct4,Klf4 at the blastocyst stage and Nanog at the 4-cell stage).Moreover,the expression level of the apoptosis-related gene Caspase-3 was significantly lower in the Scriptaid-treated SCNT embryos compared with the control SCNT embryos at the 4-cell and blastocyst stages.In conclusion,these results indicate that Scriptaid treatment improves the development and nuclear reprogramming of porcine SCNT embryos.     

Transformation of plant young proembryos by electroporation

It is first reported that plant young proembryos expressed exogenous reporter genes by electroporation. Young proembryos with 8–32 cells and globular proembryos with 250–400 cells could be isolated by enzymatic maceration combined with microdissection. After electroporation withGUS orGFP genes, the proembryos were cultured for 1–2 d in KM8p medium. At the field strength of electroporation 500–1500 V/cm, blue reaction of GUS or green fluorescence of GFP could be observed in the proembryos. The highest transient expression frequency of young proembryos (2.2%) was obtained at the field strength of 750 V/cm, whereas the highest frequency of globular proembryos (5.9%) was obtained at the field strength of 1 250 V/cm. Taking the proportion of transformed cells in the whole cells of proembryos as efficient transformation frequency, the efficient transformation frequency of the young proembryos was 7 times that of the globular proembryos.     

菠菜SoNAC转录因子家族的全基因组鉴定与分析

采用生物信息学分析方法,从菠菜基因组中筛选鉴定了57个菠菜NAC转录因子,并对其基因结构、编码蛋白和系统进化进行了分析;通过荧光定量聚合酶链式反应qRT-PCR分析,研究了高温和盐处理后菠菜叶片中NAC基因的表达模式.研究结果显示:菠菜NAC转录因子可以被归入2组17个亚组,GroupⅠ包含10个亚组,GroupⅡ包含...     

人WDR79基因启动子表达载体的构建及鉴定

人WDR79是一种重要的支架蛋白,在端粒酶组装、卡哈尔体(Cahar body)形成和DNA损伤修复等过程中发挥着重要作用。采用PCR扩增技术获得人WDR79基因启动子上游DNA序列,构建人WDR79基因启动子荧光素酶报告基因载体pGL3-WDR79-promoter;通过双酶切、琼脂糖凝胶电泳、DNA测序和荧光素酶活性测定等实验手段,验证质粒pGL3-WDR79-promoter构建的正确性及其表达活性。本研究结果为进一步探讨人WDR79基因表达的调控机制奠定实验基础。     

Effect of monoclonal antibody to bull sperm sp18 on murine fertilization and embryos development

Western blot analysis revealed that one IgG1 monoclonal antibody (mAb) to sp18 family membrane proteins (Mr. 14, 16 and 18 ku) of bovine sperm reacted faintly with protein bands of 14, 18, 22, 30 and 60 ku (reducing) in samples of mouse sperm. The mAb also reacted to protein of egg lysozyme. Using a laser confocal microscope, indirect immunofluorescence (IIP) showed that the sp18 antigens were present in the posterior head of murine sperm. In murine in vitro fertilization (IVF) and embryo development trails, a total of 426 oocytes from C57BL/6 and F1 hybrid strain (CD1 × C57BL/6 cross) of 12 female mice were used in 3 independent trails. After preincubating capacitated sperm with 182 μg/mL of sp18 mAb in the modified TYH IVF medium for 15-20 min, cumulus-oocyte-complexes were introduced. The fertilization rate in sp18 mAb groups was 77.1 %, which was not significantly (P > 0.05) different from the nonspecific mouse IgG (79.2%) and non-IgG (80.3 %) control groups. Fertilized oocytes had been continuously cultured in modifed CZB medium. 100%, 100% and 97.9% of 1-cell embryos developed to 2-cell stage in sp18 mAb, nonspecific mouse IgG and non-IgG group 30 h after the start of fertilization, respectively. In the nonspecific mouse IgG and non-IgG groups, 64.1 % and 64.3% of embryos developed to the 4-cell stage, respectively, but all developing eggs in sp18 mAb groups arrested development in vitro at 2-cell stage. After zonae of 2-cell blocked embryos were enzy-matically removed with 0.5% pronase, detection of sp18 antigens by IIF indicated that the fluorescence scattered on two embryonic cells. For embryos fertilized in vivo and co-cultured with 182 μg/mL sp18 mAb, the numbers of 1-cell embryos reaching the 2-cell and 4-cell stage were 95. 2% and 70. 5%, which were not significantly (P>0.05) different from the control group (92.9% and 77.9%). These results indicate that the sp18 antigens on posterior head of mouse sperm were incorporated into the egg plasma membrane during fertilization, and played an active role in development of murine preimplanta-tion embryo.     

广西红树林区有孔团水虱的卵子发生

摸清有孔团水虱的繁殖特性,对团水虱的防控和消杀,以及保护红树林生态系统具有重要意义。本研究采用组织切片技术,研究广西红树林区有孔团水虱的卵子发生过程,探讨其繁殖期时间。结果表明,有孔团水虱的卵子发生过程可分为4个时期,分别是卵原细胞、无卵黄卵母细胞、卵黄发生卵母细胞和成熟期卵母细胞。其中：无卵黄卵母细胞又可分为前期、中期和后期3个亚期;卵黄发生卵母细胞可分为卵黄开始沉积时相和卵黄充满时相2个亚期。受精后发育的胚胎存在于母体内。周年调查表明：有孔团水虱雌性体内全年存在着各时期的卵母细胞或胚胎;2-3月无成熟期卵母细胞或胚胎出现。结合全年各期卵母细胞分布以及胚胎和幼体情况判断,广西红树林区有孔团水虱的繁殖期为每年的4月至次年的1月。     

Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter

The green fluorescent protein (GFP) gene from the jellyfishAequorea victoria as a vital reporter for gene expression in plants is considered to have several advantages over other reporter genes. The pBIN35S-mGFP4 plasmid DNA has been introduced into cotton embryos by the pollen-tube pathway method. A transformed seedling has been verified according to its GFP-related fluorescence and Southern blotting analysis. The results provided direct and convincing facts in cytology and molecular biology for the pollen-tube pathway method, an efficient transformation technique used in plants.     

pEGFP-C1-MCH真核表达载体的构建及其在HEK293 细胞系中的表达

目的:构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型.方法:提取脑组织总RNA,反转录为cDNA,参照Genbank中提供的序列设计引物扩增MCH基因全长.再将该基因全长cDNA克隆至质粒pEGFP-C1,经菌落PCR筛选及双酶切和DNA测序鉴定,成功构建了含有目的基因MCH的重组质粒pEGFP-C1-MCH.并利用脂质体2000介导其转染HEK293细胞,用荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达.结果:克隆的pEGFP-C1-MCH质粒序列中的MCH与Gen Bank相符;细胞转染72 h后,转染成功的细胞在荧光显微镜下表达较强的绿色荧光,MCH基因稳定表达.结论:pEGFP-C1-MCH真核表达载体的构建及其在HEK293细胞中的稳定表达,为研究MCH基因在能量代谢中的功能及作用机制提供了实验模型.     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

基于数据库分析FOXG1在非小细胞肺癌中的表达和临床意义及稳定过表达FOXG1肺癌细胞株A549的构建

通过TCGA数据库分析FOXG1在非小细胞肺癌中的表达及预后相关性，建立稳定过表达人源FOXG1基因的肺癌细胞株A549。利用TCGA数据库中下载基因表达数据和临床信息，分析FOXG1在非小细胞肺癌和正常组织的表达差异、FOXG1表达水平与临床病理特征及生存预后的相关性并进行基因集富集分析。通过HEK-293T包装慢病毒表达载体，收集病毒上清液侵染A549细胞,嘌呤霉素筛选稳定过表达FOXG1的A549细胞株。细胞核染色鉴定外源FOXG1表达定位，Western blot检测外源FOXG1的表达情况。结果发现FOXG1在非小细胞肺癌组织中高表达，且FOXG1高表达能够降低患者总体生存率。FOXG1的表达水平与患者年龄相关，与性别，分级以及TMN分期无关；细胞周期、P53、Notch等信号通路在高表达FOXG1的非小细胞肺癌组织中被激活；慢病毒表达载体共转染HEK-293T细胞成功；病毒上清液侵染A549细胞，24 h后可见绿色荧光表达，72 h后对照组空载体病毒颗粒侵染效率高达80%左右，实验组过表达FOXG1病毒颗粒侵染效率为50%~60%；经嘌呤霉素筛选培养后，对照组和实验组荧光效率均达到90%以上；细胞核染色外源FOXG1基因定位在细胞核中，Western blot结果显示外源FOXG1在细胞中正确表达。因此可以认为FOXG1基因在非小细胞肺癌患者中高表达，其表达水平与患者总体预后有关且稳定表达FOXG1的A549肺癌细胞株构建成功。