Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ-specific expression and genetic analysis

Summary The distribution of the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family (Gpc) in the maize genome was investigated; a genetic variant of glyceraldehyde-3-phosphate dehydrogenase activity is also described. Restriction fragment length polymorphism analysis of an F₂ population shows that the variant is not linked to the three known Gpc genes. However, this trait is linked to one of two genomic DNA fragments that hybridize to a fragment of the Gpc3 coding region, implying the existence of a fourth Gpc gene. Antibodies and cDNA clones were used to investigate the organ-specific expression of the Gpc genes. Results were compared with the expression of the alcohol dehydrogenase 1 (Adh1) gene. RNA and protein levels were examined in seedling roots and shoots, as well as the leaves, developing endosperm and embryo, and the aleurone. In general, it was found that Gpc3 expression behaves in parallel with Adh1 in these organs, and protein levels closely parallel that of RNA for each gene examined. Both Gpc3 and Adh1 show a marked increase in expression during endosperm development, reaching a maximum 15 days after pollination, but no expression is detected in the leaf. Gpc1 expression is similar to that of Gpc2, with an overall decrease in the level of RNA during endosperm development. This expression is discussed in terms of the common sequences found upstream of genes expressed in the developing maize seed.     

Structure of the HOM2 gene of Saccharomyces cerevisiae and regulation of its expression

Summary In Saccharomyces cerevisiae the HOM2 gene encodes aspartic semi-aldehyde dehydrogenase (ASA DH). The synthesis of this enzyme had been shown to be derepressed by growth in the presence of high concentrations of methionine. In the present work we have cloned and sequenced the HOM2 gene and found that the promoter region of this gene bears one copy of the consensus sequence for general control of amino acid synthesis. This prompted us to study the regulation of the expression of the HOM2 gene. We have found that ASA DH is the first reported enzyme of the related threonine and methionine pathway to be regulated by the general control of amino acid synthesis.     

高粱高亲和硝酸盐转运蛋白NRT2/3基因家族鉴定、表达与DNA变异分析

硝态氮是作物吸收无机氮素的主要形态,硝酸盐转运蛋白2(nitrate transporter 2,NRT2)作为高亲和性的转运蛋白,以硝酸盐作为特异性底物,在可利用的硝酸盐受限时,高亲和性转运系统被激活,在硝酸盐吸收、转运过程中发挥着重要作用。大多数NRT2不能单独转运硝酸盐,需在硝酸盐同化相关蛋白2(nitrate assimilation related protein 2,NAR2)的协助下才能完成硝酸盐的吸收或转运。作物氮利用效率受环境条件影响,品种间存在差异,因此培育高氮素利用效率品种有重大意义。高粱(Sorghum bicolor)具有耐贫瘠特性,对土壤中的氮素吸收和利用效率较高。本研究结合高粱基因组数据库对NRT2/3基因家族成员基因结构、染色体定位、理化性质、二级结构与跨膜结构域、信号肽与亚细胞定位、启动子区顺式作用元件、系统进化、单核苷酸多态性(single nucleotide polymorphism,SNP)的识别与注释及选择压力进行了全面分析。通过生物信息学分析,筛选出5个NRT2s(命名为SbNRT2-1a、2-1b、SbNRT2-2–4)基因和2个NAR2s(SbNRT3-1–2)基因,较谷子略少。分布在3条染色体上,分为4个亚家族,同一亚族中基因结构高度相似;高粱NRT2/3亲水性平均值均为正值,表明均为疏水性蛋白;α-螺旋和无规则卷曲占二级结构总量的比例大于70%;亚细胞定位均在质膜上,其中NRT2s蛋白不含信号肽,NRT3s蛋白含信号肽;进一步对其跨膜结构域进行分析,发现NRT2s家族成员跨膜结构域个数均大于10个,而NRT3s家族成员跨膜结构域个数为2个;高粱与玉米(Zea mays)NRT2/3s的共线性较好;蛋白结构域显示存在MFS_1和NAR2蛋白结构域,可执行高亲和力硝酸盐转运;系统进化树分析可知,高粱与玉米和谷子的NRT2/3基因亲缘关系更近;基因启动子顺式作用元件分析发现,SbNRT2/3基因的启动子区均具有数个植物激素和逆境应答元件,可以响应高粱生长和环境变化;基因表达热图显示低氮条件下在根诱导表达的是SbNRT2-1a、SbNRT2-1b和SbNRT3-1,推测可在高粱根部表达并调控对硝酸盐的吸收或转运过程。在SbNRT2-4和SbNRT2-1a等发现多个非同义SNP变异;选择压力分析表明,高粱NRT2/3基因家族在进化过程中受纯化选择作用。SbNRT2/3基因表达及蚜虫侵染影响与基因在不同组织中的表达分析结果一致,SbNRT2-1b和SbNRT3-1在感染蚜虫品系5-27sug根部表达显著,高粱蚜虫侵染叶片显著降低了SbNRT2-3、SbNRT2-4和SbNRT3-2的表达水平。本研究初步对高粱全基因组NRT2/3基因家族进行鉴定、表达与DNA变异分析,为高粱氮高效研究提供了基础。     

纤枝短月藓BeLEA2基因的克隆及表达分析

为探究纤枝短月藓LEA2基因的结构和表达特征,该研究以纤枝短月藓为材料,首次利用PCR克隆技术得到纤枝短月藓BeLEA2基因序列,并对该基因进行分析。结果表明:(1)该基因序列中含有2个外显子和1个内含子,其开放阅读框(ORF)为456 bp,编码151个氨基酸,预测其相对分子质量为16515.96 Da。(2)将纤枝短月藓与其他植物LEA2基因氨基酸序列进行比对,构建系统进化树,结果显示纤枝短月藓与小立碗藓的亲缘关系最近。(3)利用HiTail-PCR技术克隆获得1072 bp的BeLEA2启动子序列,用PlantCARE在线工具对该启动子的顺式作用元件进行预测,结果表明该启动子除了含有核心启动子元件TATA-box和CAAT-box外,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他顺式元件。(4)实时荧光定量PCR分析表明,BeLEA2基因在纤枝短月藓不同发育时期和不同组织中都有表达,且对脱水胁迫有响应。以上结果为进一步探究LEA2基因在苔藓植物中的功能及作用机制奠定了基础。     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Flrt2 and Flrt3 have overlapping and non-overlapping expression during craniofacial development

Craniofacial morphogenesis is a complex multi-step process that involves numerous biological processes to coordinate the growth, proliferation, migration, and subsequent differentiation of the cranial neural crest cells. Members of the Fibronectin Leucine-Rich Transmembrane (Flrt) gene family have been previously reported to be widely expressed in the developing embryo. We mapped the expression of Flrt2 and Flrt3 at critical stages of craniofacial development and found that, during early craniofacial development, Flrt2 was highly expressed initially in the cranial neural crest cells and Flrt3 in the midbrain. Later both genes were expressed in the developing pharyngeal region. Flrt2 expression predominated in the neural crest-derived mesenchyme in the medial aspect of the developing frontonasal region in close relationships with the expression of Fgfr2, Shh, and Msx1, three genes shown previously to play critical roles in craniofacial development. Flrt2 was also present in the vomero-nasal organ, mandibular primodia, and the posterior aspects of the unfused and fused secondary palatal shelves. Flrt3, however, had a more restrictive expression, being present in the mesenchyme underlying the ectoderm of the medial nasal process and in the mandibular primordium and in regions undergoing outgrowth, in a pattern that overlapped with Bmp4 expression. Both Flrt2 and Flrt3 were later found to be present at sites of epithelial–mesenchymal interactions such as the developing tooth buds, hair follicles, and eye. Together the data suggested important roles for Flrt2 and Flrt3 in mediating events such as NCC migration, chondrogenesis and epithelial–mesenchymal interactions during craniofacial development.     

Cloning and expression analysis of putative glyceraldehyde-3-phosphate dehydrogenase genes in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were cloned from Pilobolus crystallinus with degenerated primers designed from conserved sequences in many GAPDHs. P. crystallinus had three gapdh homologue genes, named pcgapdh1, pcgapdh2, and pcgapdh3. Deduced amino acid sequences for PCGAPDH1, PCGAPDH2, and PCGAPDH3 showed highest similarity with GPD3, GPD1, and GPD2, respectively, of Mucor circinelloides, indicating that these three gapdh genes had diverged before Pilobolus and Mucor were separated. The expression patterns of the gapdh genes, however, were quite different between P. crystallinus and M. circinelloides. All the three pcgapdh genes were expressed, and the expression of pcgapdh2 was suppressed by glucose and sodium acetate. These results indicate that the function of these orthologous genes was changed after Pilobolus and Mucor were separated.     

白及小分子热激蛋白BsHsp17.3基因的克隆与表达分析

为了探索人工栽培白及的适宜条件,该研究以湖北省十堰市野生白及为对象,采用同源克隆和3'RACE技术,从白及(Bletilla striata)中获得与热激蛋白合成有关的BsHsp17.3基因,并分析BsHsp17.3基因对不同胁迫的响应。结果表明:BsHsp17.3基因开放阅读框长度为453 bp,编码150个氨基酸;蛋白的分子量为17.42 kD,等电点为6.33。进化树分析表明BsHSP17.3蛋白与同为兰科的铁皮石斛进化关系较近,同在一分支上。半定量RT-PCR分析显示BsHsp17.3基因在白及根、叶、鳞茎及花组织中的表达具有特异性,且BsHsp17.3基因在叶中的表达量较高,在鳞茎及花中不表达。实时荧光定量PCR检测显示BsHsp17.3对非生物胁迫高温、低温具有明显应答反应,20%PEG模拟干旱胁迫不诱导该基因表达,推测该基因在白及防止倒苗过程中可能发挥一定作用。     

甜荞FeFUL2基因的克隆与表达分析

为了探索甜荞FUL同源基因参与花与籽粒发育调控的分子机制,该文采用同源克隆的方法从甜荞(Fagopyrum esculentum)长花柱和长雄蕊突变体(lpls)中克隆到1个长837 bp的FeFUL2基因(GenBank登录号为MG779493.1),其包含长690 bp的完整开放阅读框,编码1个由229个氨基酸残基组成的MADS-box转录因子。通过对FeFUL2进行分子系统发生、同源蛋白比对与转录因子结构分析,结果显示FeFUL2与核心真双子叶植物AP1/FUL亚家族转录因子中的euFUL进化系聚于1个进化分支,属甜荞euFUL型MADS-box转录因子,且包含1个57个氨基酸残基长的高度保守的MADS结构域、1个69个氨基酸残基长的次级保守的K结构域,其C末端转录激活区在序列长度和氨基酸残基组成上与其他euFUL型转录因子差异较大,但仍含有2个euFUL型转录因子特有的保守基元:FUL motif和paleo AP1 motif。用qPCR检测基因表达的组织特异性显示:FeFUL2基因在甜荞lpls突变体的根、茎、叶、花被片、雄蕊、雌蕊和发育4 d的幼果中均有表达,但其在花被片中表达量极显著高于该基因在其他器官中的表达量(LSD,P0.01)。综合转录因子的结构与基因的表达模式推测,FeFUL2基因与其他euFUL型基因的功能可能存在一定差异,其在花发育过程中可能主要参与甜荞花被片的发育调控。     

热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2的功能评价

【目的】热带假丝酵母是发酵法生产二元酸的重要工业菌株,具有较高的ω-氧化活性。脂肪醛脱氢酶在ω-氧化途径中起重要作用,催化脂肪醛生成脂肪酸,但其具体催化功能及对细胞生理影响还未被系统研究。本文通过删除脂肪醛脱氢酶基因CtAld1和CtAld2鉴定了其在ω-氧化途径中的功能。【方法】通过基因组信息挖掘获得热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2序列,在此基础上,通过同源重组敲除CtAld1和CtAld2基因。考察突变株的生长和胞内脂肪醛脱氢酶活性变化,并评价CtAld1和CtAld2基因敲除对细胞二元酸合成能力的影响。【结果】分别获得了热带假丝酵母突变株XZX-1(ΔCtAld1/ΔCtAld1)、XZX-2(ΔCtAld2/ΔCtAld2)和XZX-12(ΔCtAld1/ΔCtAld1,ΔCtAld2/ΔCtAld2)。在以十二烷为唯一碳源的培养基中,敲除CtAld2基因显著抑制细胞的生长,胞内脂肪醛脱氢酶活性降低为出发菌株的30%;敲除CtAld1基因尽管会使细胞损失一部分醛脱氢酶活性,但能够一定程度地提升细胞在十二烷中的生长性能。敲除CtAld1或CtAld2会降低菌株二元酸产量,组合敲除CtAld1和CtAld2严重削弱菌株十二碳二元酸的合成能力。【结论】CtAld2对热带假丝酵母细胞的生长和十二碳二元酸的合成具有重要作用,缺失CtAld1或CtAld2基因降低细胞的二元酸合成能力。CtAld1和CtAld2可作为热带假丝酵母ω-氧化途径代谢工程改造的潜在靶点。     

夏枯草PvDXS基因的克隆和表达分析

该研究在夏枯草转录组测序的基础上设计特异引物,采用逆转录PCR技术获得该基因的全长核苷酸序列,并进行生物信息学分析,采用qRT-PCR法分析PvDXS在夏枯草不同组织及不同外源性物质诱导下的表达量。结果表明:克隆得到的PvDXS基因开放阅读框2 181 bp,编码726个氨基酸,理论分子量为78 040.47 D,等电点为6.75,PvDXS蛋白具有Transketolase_C结构域和Transket_pyr结构域,系统进化树结果表明,PvDXS蛋白与丹参、长春花的DXS(SmDXS2、CrDXS2)亲缘关系较近,推测PvDXS属于第Ⅱ类DXS蛋白。qRT-PCR分析表明,PvDXS基因在叶中表达量高于果穗及茎。对果穗施加7种外源性物质处理24 h后,GA₃处理组该基因表达量升高,其他6种外源性物质处理后表达量均降低,其中CaCl₂、SNP、SA处理后该基因的表达量显著降低。PvDXS基因在不同组织中表达量差异较大,且受外源物质诱导表达。这为进一步研究PvDXS基因对夏枯草萜类成分合成途径中的功能及表达调控奠定基础。     

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(M) phenotype in Drosophila ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.