非编码的mRNA

非编码的mRNA是近年发现的一类不含典型ORF的mRNA.目前已发现或克隆的这类基因主要有：H19基因,XIST基因,XLSIRT基因,His-1基因,bic基因,rox1和rox2基因等.它们与胚胎发育,肿瘤发生及X染色体失活密切相关.     

蔗茅EfMYB1基因的克隆与表达分析

为了充分利用蔗茅(Erianthus fulvus)野生资源,挖掘其优良的抗性基因,丰富转基因甘蔗育种候选基因库,该研究结合蔗茅转录组数据,以蔗茅99 1无性系为试验材料,利用RT PCR技术克隆蔗茅MYB基因,并对其进行生物信息学分析及胁迫表达分析,以解析蔗茅的耐寒机理,为转基因甘蔗育种奠定理论基础。结果表明：(1)成功克隆得到一个蔗茅MYB基因,命名为EfMYB1基因(登录号ON586646)。(2)生物信息学分析表明,EfMYB1基因全长1 000 bp,ORF为759 bp,编码251个氨基酸;编码蛋白具有一个保守的SANT结构域,无跨膜结构和信号肽,有多个磷酸化位点;二级结构与三级结构主要以α螺旋和无规则卷曲为主;与南荻相似性最高,遗传距离最近。(3)qRT PCR分析结果发现,EfMYB1基因在蔗茅根和叶组织中的相对表达量随低温胁迫时间的持续而逐渐显著上调,并于胁迫72 h时达到最大值,而在茎中的表达则几乎没有变化;茉莉酸甲酯胁迫下,EfMYB1基因的相对表达量呈先升高后降低的趋势,且在处理6 h时达到最高值;脱落酸胁迫下EfMYB1基因的表达水平较0 h时极显著降低。研究认为,EfMYB1基因属于低温胁迫响应基因,可能参与蔗茅低温胁迫下的应答反应。     

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.     

不同代次牛成肌细胞的诱导分化及相关基因的表达分析

利用初生荷斯坦牛的成肌细胞,在不同代次以含体积分数为2%马血清的DMEM进行诱导分化,在之后0、2、4、6、8、10 d观察细胞的形态变化并收集细胞提取总RNA,检测成肌相关基因的表达情况,为进一步研究牛肌肉发生过程及相关基因的表达调控提供依据。同时利用实时荧光定量PCR分别检测肌性相关基因MyoD(生肌决定因子)、MyoG(肌细胞生成素)以及非肌性相关基因A-FABP(脂肪细胞型脂肪酸结合蛋白)表达水平的变化。研究表明：①各代细胞在诱导培养4 d后开始有肌管形成,其后越来越多,8 d时达高峰。②成肌细胞向成熟肌细胞分化过程中,MyoD、MyoG、A-FABP基因都呈先上升然后下降的趋势。③高代次成肌细胞比低代次细胞增殖慢,而且在诱导分化后,肌管的数目明显变少; MyoD、MyoG、A-FABP随着代次的升高而下降。故推断MyoD、MyoG以及A-FABP基因会在成肌分化开始后的不同阶段被激活,从而发挥不同的调控作用,而且随着传代次数的增加成肌细胞的分化能力减弱。     

线虫基因显微注射和整合技术——设备、操作与指南

秀丽线虫(Caenorhabditis elegans)是研究动物遗传、个体发育和行为活动的重要模式生物,其主要优点是能够研究从分子细胞水平到整体系统水平的相关生命活动的作用机理．其中基因显微注射和整合是该领域的核心技术,广泛应用于研究线虫的基因表达、功能和基因间的相互作用等．显微注射技术使用尖端开口直径为微米级的玻璃微管,将所研究的目的DNA注射进线虫的性腺．注射的DNA被成熟的卵细胞吸收,以染色体外遗传物质的形式存在．但这种形式并不能稳定遗传,可采用基因整合技术将外源基因整合到染色体上,得到稳定遗传的线虫种系．显微注射是一项精细的实验技术,影响实验成功与否的因素较多,实际操作需要有一定的经验．在实践过程中逐步改进和完善了这项技术,在此主要介绍线虫显微注射技术的操作过程、创新方法以及注意细节．     

Anaerobic bacteria and antibiotics: What kind of unexpected resistance could I find in my laboratory tomorrow?

The purpose of this article is to set out some important considerations on the main emerging antibiotic resistance patterns among anaerobic bacteria. The first point concerns the Bacteroides fragilis group and its resistance to the combination of β-lactam+β-lactamase inhibitor. When there is overproduction of cephalosporinase, it results in increased resistance to the β-lactams while maintaining susceptibility to β-lactams/β-lactamase inhibitor combinations. However, if another resistance mechanism is added, such as a loss of porin, resistances to β-lactam+β-lactamase inhibitor combinations may occur. The second point is resistance to metronidazole occurring due to nim genes. PCR detection of nim genes alone is not sufficient for predicting resistance to metronidazole; actual MIC determinations are required. Therefore, it can be assumed that other resistance mechanisms can also be involved. Although metronidazole resistance remains rare for the B. fragilis group, it has nevertheless been detected worldwide and also been observed spreading to other species. In some cases where there is only a decreased susceptibility, clinical failures may occur. The last point concerns resistance of Clostridium species to glycopeptides and lipopeptides. Low levels of resistance have been detected with these antibiotics. Van genes have been detected not only in clostridia but also in other species. In conclusion, antibiotic resistance involves different mechanisms and affects many anaerobic species and is spreading worldwide. This demonstrates the need to continue with antibiotic resistance testing and surveys in anaerobic bacteria.     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Inheritance and segregation of exogenous genes in transgenic cotton

Three transgenic cotton varieties (lines) were chosen for the study of inheritance and segregation of foreign Bt (Bacillus thuringiensis toxin) andtfdA genes in cotton. The transformed cotton varieties CCRI 30 and NewCott 33B expressing the BtcryIA gene, and cotton line TFD expressing thetfdA gene were crossed with CCRI 19, CCRI 12 and Lumian 6. The results confirm inheritance and segregation of (i) the exogenous Bt gene in transgenic CCRI 30 and NewCott 33B, governing resistance to bollworm, and (ii) the exogenoustfdA gene in transgenic TFD, governing resistance to the herbicide 2,4-D. Both resistance characters were governed by a single dominant nuclear gene, and were not affected by cytoplasm. Our data support the conclusion that foreign traits encoded by single genes are inherited and expressed in Mendelian fashion in cotton. Our results also indicate that a practical backcross breeding program could be used to develop cotton cultivars combining one or more resistance traits from foreign and native gene sources.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

转双价抗虫基因大白菜新种质的获得及其抗虫性分析

采用超声波辅助花粉介导方法,将双价抗虫基因BmkIT-Chitinase导入早熟型大白菜自交系20-19-3,最终获得了5个转基因大白菜优良自交系纯合株系Z1-5、Z2-7、Z9-6、Z11-6和Z20-13;以转基因大白菜株系和非转基因对照植株为材料,对BmkIT-Chitinase基因在大白菜中的遗传规律、基因表达及抗虫性进行进一步分析。结果显示:(1)转化株后代多代(T_1~T_4)PCR、Southern blotting等分子跟踪检测表明,目的基因已成功导入受体植株,且能够稳定遗传;用该转基因方法对大白菜进行基因转化所获得的转基因植株分析显示,外源基因多数以多拷贝形式整合于核基因组,少部分外源基因以单拷贝形式整合。(2)Elisa分析结果证明,所导入的外源基因可高效表达,T4代株系新鲜叶片中表达产物量最高达到0.069μg·g~(-1)左右。(3)转基因株系田间抗虫性统计分析表明,转化株系与对照在抗虫性方面有显著差异,其对小菜蛾及菜青虫抗性普遍提高2~3级。研究认为,转BmkIT-Chitinase基因大白菜中BmkIT-Chitinase基因的表达可有效提高大白菜的抗虫性。     

毛尖紫萼藓抗旱相关基因Gp-LEA的克隆与表达分析

以毛尖紫萼藓干旱cDNA文库中获得的一段与LEA基因同源性较高的EST序列为基础,采用RACE技术分离该基因cDNA全长序列,命名为Gp-LEA。Gp-LEA基因的cDNA全长814bp,开放阅读框456bp,编码含151个氨基酸蛋白质。生物信息学分析结果显示,Gp-LEA蛋白为稳定蛋白,分子质量为16.612kD,理论等电点（pI）为5.06,含有LEA₂功能结构域,不属于跨膜蛋白且不存在信号肽。系统发生分析表明,Gp-LEA基因编码蛋白与花旗松LEA蛋白亲缘关系最近。荧光定量PCR分析显示,Gp-LEA基因在复水和快速干旱模式下均能表达。推测Gp-LEA基因在毛尖紫萼藓的复水和干旱过程中起着重要作用。     

hpc2研究进展

生物个体的胚胎发育以及细胞的增殖、分化,都同时受到多种基因的严格调控,PcG基因家族就是一类重要的发育相关基因.而hPc2基因是人PcG基因家族中的一个重要成员,其编码的HPC2蛋白,不仅可以和HPH、BMI-1以及RING1等其他人类PcG蛋白结合形成HPC/HPH PcG复合体,以蛋白复合体的形式参与对homeotic基因的表达抑制,以维持机体的正常发育以及细胞的增殖和定向分化,还发现它能与其他多种蛋白质相结合,提示HPC2可能具有多种功能.因此,对hPc2的深入研究不仅有助于进一步阐明PcG基因家族的作用机理,扩展人们对基因表达调控的认识,还有助于发现PcG基因家族与其他信号转导通路的联系,更好地理解细胞信号网络系统.     

火鹤AaSERK基因克隆及其在体细胞胚胎发生中的表达分析

为了探讨体细胞胚胎发生相关类受体蛋白激酶(SERK)基因在火鹤体细胞胚胎发生中的作用,以火鹤胚性愈伤组织为材料,利用RT-PCR结合RACE技术,克隆了火鹤体细胞胚胎发生相关类受体蛋白激酶基因(AaSERK),通过实时荧光定量PCR(Real time-PCR)技术分析了AaSERK的相对表达量。结果显示:(1)该基因全长为1 949bp,包含1 866bp开放阅读框,编码蛋白由622个氨基酸组成。(2)预测该基因具有SERK家族典型的结构域:1个信号肽、1个亮氨酸拉链结构域、5个富亮氨酸重复序列结构域、1个SPP基序、1个跨膜结构域、含11个亚区的激酶结构域、1个C端结构域。DNAMAN分析显示,该基因编码的氨基酸序列与其它植物的一致性达到65%~89%。(3)在离体诱导体细胞胚胎发生的各阶段中,AaSERK基因在诱导阶段及发育培养阶段微弱表达或者几乎不表达,在继代培养第30天的胚性愈伤组织中表达量最高。推测该基因可以作为火鹤体细胞胚胎发生的一个标记基因。     

Sub-cellular Localisation of the White/Scarlet ABC Transporter to Pigment Granule Membranes Within the Compound Eye of Drosophila Melanogaster

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.