Analysis ofcis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea

Cis-regulatory elements involved in the activation of the plant defense-related gene encoding chalcone synthase 1 (PsChs1) in pea (Pisum sativum L.) were examined by transient transfection, gel mobility shift assay andin vitro DNase I-footprinting analysis. Transient transfection assay revealed that a 61 bp DNA fragment spanning from –242 to –182 ofPsChs1 was required for the maximal promoter activity and possibly involved in the enhancement of elicitor-mediated activation. Nuclear isolate from elicitor-treated pea epicotyl tissues contained some factor(s) that specifically bound to this DNA fragment to form a complex with low mobility (LMC, low mobility complex) in gel mobility shift assay. DNase I-footprinting analysis of LMC revealed that among three protected regions detected in a 61 bp DNA fragment, two regions contained identical AT-rich sequence, TAAAATACT. Site directed mutation in either or both identical sequences, TAAAATACT to TGGAATACT, resulted in the reduction or loss in the ability to form LMC. Detailed analysis of 61 bp DNA fragment demonstrated that the region from –242 to –226 containing promoter-distal TAAAATACT motif was imperative for the maximal elicitor-mediated activation ofPsChs1.     

Phylogenetic relation of Epidermophyton floccosum to the species of Microsporum and Trichophyton in chitin synthase 1 (CHS1) gene sequences

The Nucleotide sequence of the chitin synthase1 (CHS1) gene of Epidermophyton floccosum, an anthrophophilic dermatophyte which is the type species of the genus Epidermophyton was analyzed to determine its phylogenetic relation to eight other dermatophyte species belonging to the genera Microsporum and Trichophyton, which were sequenced in our previous studies. A genomic DNA fragment about 620 bp in length of the CHS1 genewas amplified from E. floccosum by polymerasechain reaction (PCR) and was sequenced. The CHS1 nucleotide sequence showed more than 85% similarity with sequences derived from the other dermatophytes. Phylogenetic analyses of the sequences from E. floccosum revealed that the genus Epidermophyton may be genetically distinct from Microsporum and Trichophyton. This revised version was published online in June 2006 with corrections to the Cover Date.     

蒺藜苜蓿DGAT1基因的克隆和功能鉴定

该研究采用RT-PCR与电子克隆的方法,从蒺藜苜蓿cDNA中克隆得到2个编码二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)的基因MtDGAT1-1和MtDGAT1-2。MtDGAT1-1长1 620bp,编码539个氨基酸;MtDGAT1-2长1 524bp,编码507个氨基酸。多序列比对显示,MtDGAT1-1和MtDGAT1-2编码蛋白具有典型的植物DGAT1结构域。表达分析显示,MtDGAT1-1和MtDGAT1-2在根、茎、叶、花、种子中都有表达,在种子发育中高表达,且MtDGAT1-1于种子发育的中前期高表达,而MtDGAT1-2于种子发育的中后期高表达。酵母互补实验证实,MtDGAT1-2编码蛋白具有DGAT酶活性,能够恢复H1246的TAG合成和油体形成;而MtDGAT1-1编码蛋白不能恢复H1246的TAG合成和油体形成。     

中间锦鸡儿转录因子基因 CiNAC1的克隆及功能分析

NAC转录因子家族是植物特有的、最大的转录因子家族之一,在植物应答非生物胁迫和生长发育过程中有重要的功能。该研究通过PCR技术,克隆得到了中间锦鸡儿 CiNAC1基因1 066 bp的cDNA全长序列。生物信息学分析显示, CiNAC1基因的开放阅读框(ORF)为921 bp,编码306个氨基酸,推导的蛋白分子量为34.57 kD,等电点为8.35,是一种亲水性蛋白,N端具有保守的NAM结构域,具有26个磷酸化位点和7个糖基化位点。实时荧光定量PCR检测显示,中间锦鸡儿 CiNAC1基因表达受干旱、高盐、脱水、高pH诱导;亚细胞定位发现CiNAC1定位到细胞核中,这与它作为转录因子的功能是一致的;转CiNAC1基因拟南芥株系侧根数目显著多于野生型,根长也明显比野生型长。研究认为, CiNAC1基因可能与中间锦鸡儿响应逆境胁迫机制有关。     

Complete Structural Organization of the Human α1(V) Collagen Gene (COL5A1): Divergence from the Conserved Organization of Other Characterized Fibrillar Collagen Genes

Genes that encode the vertebrate fibrillar collagen types I–III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization ofCOL5A1,the human gene that encodes the α1 chain of fibrillar collagen type V. The structure ofCOL5A1is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I–III.COL5A1has 66 exons, which is greater than the number of exons found in the genes for collagen types I–III. The increased number of exons is partly due to the increased size of the pro-α1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I–III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region ofCOL5A1compared to the organization of the triple-helix coding regions of the genes for collagen types I–III. Of particular interest is the increase of 54 bp exons in this region ofCOL5A1,strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure ofCOL5A1to the highly conserved structure of the genes of collagen types I–III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes.     

The internal transcribed spacer 1 (ITS-1), a controversial marker for the genetic diversity of Trypanosoma evansi

Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338–342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3–3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8–14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

A phylogenetic study ofPolygonum sect.Tovara (Polygonaceae) based on ITS sequences of nuclear ribosomal DNA

Polygonum sect.Tovara comprises three morphologically very similar species;P. virginianum,P. filiforme, andP. neofiliforme. Sequences of internal transcribed spacers (ITSs) of nuclear ribosomal DNA of these were determined to examine phylogenetic relationships and the levels of differentiation among them. The size of ITS 1 was 241 bp inP. filiforme andP. neofiliforme, and 242 bp inP. virginianum. The size of ITS 2 was 243 bp, and that of the 5.8S rRNA coding region was 163 bp. The ITS sequences clearly separate North AmericanP. virginianum from the eastern Asian species. Nucleotide divergence between them ranges from 3.3% to 3.8% for ITS 1 and from 9.3% to 10.7% for ITS 2. The molecular data also revealed that two eastern Asian species are closely related but should be treated as distinct species.     

长春花丙二烯氧化物合酶基因的克隆与表达

利用RT-PCR和RACE相结合的方法,从长春花中克隆了丙二烯氧化物合酶(AOS)基因。结果显示:长春花AOS基因(CrAOS)cDNA全长为2 118bp,包括5′和3′非翻译区,polyA尾和一个长1 638bp的开放阅读框,其基因组中不含内含子;CrAOS基因编码的蛋白含545个氨基酸。多重比对表明CrAOS蛋白与其他的AOS蛋白具有较高的相似性,CrAOS蛋白序列中含有AOS家族应有的保守氨基酸残基。Southern杂交表明:CrAOS基因在长春花中为低拷贝。qRT-PCR结果显示:CrAOS在各个组织均有表达但表达量存在差异,在老叶中最高,在幼花中表达最低。对长春花幼苗进行不同处理,结果表明:伤害、低温、甲基茉莉酸、乙烯利处理等可使CrAOS基因表达量显著提高,水杨酸处理对基因表达影响不大。     

Mapping, Expression, and Association Study of the Bovine PSMC1 Gene

The 26S ATP-dependent protease is composed of a 20S catalytic proteasome and two PA700 regulatory modules; it plays a central role in many regulatory pathways, such as cell cycle regulation, differentiation, and apoptosis. The PA700 complex is composed of multiple subunits, including at least six related ATPases and approximately 15 non-ATPase polypeptides. PSMC1 (proteasome 26S subunit, ATPase, 1) is one of these ATPases. In this study, we amplified a fragment of 507 bp from intron 9 of the bovine PSMC1 gene and found a SNP (G/A) at position 216 in the PCR fragment. Genotyping of 138 animals from four beef breeds revealed that the average frequency for allele A (G-base) was 0.4271 (0.3269–0.5517); for allele B (A-base) it was 0.5729 (0.4483–0.6731). This SNP is significantly associated with average daily feed intake (P < 0.01), average daily gain, finishing average daily gain, body length, ratio of feed to meat, backfat thickness, and loin-muscle area (P < 0.05). Our experimental data showed that animals with an AA genotype have a significantly lower food intake, grow faster, are longer in the body, and have less backfat and bigger loin muscle; hence, their ratio of feed to meat is significantly lower. We believe that the PSMC1 SNP is a potential candidate marker for marker-assisted selection in these traits. We also found that the bovine PSMC1 gene was expressed mainly in lung, testis, and spleen. In addition, we mapped the bovine PSMC1 gene on BTA10 by an RH mapping method. The sequences of the 140 bp fragment (accession no. DQ111992) and the 507 bp fragment (accession no. DQ139315) have been submitted to GenBank     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Allele frequency of two intragenic microsatellite loci of SEL1L gene in Northern Italian population

Two cytosine-adenine (CA) repeats CAR/CAL and RepIN20 occur in the human SEL1L gene, which is regarded as a candidate gene for insulin-dependent diabetes mellitus (IDDM) and Grave's disease. We have characterized these repeats to determine if they might serve as effective microsatellite markers for linkage analysis to clarify whether SEL1L gene plays a role in the pathogenesis of these autoimmune diseases. The allele frequencies and average heterozygosity of the microsatellite repeats were analysed in 94 DNA samples from peripheral blood mononuclear (PBMC) cells from adults of Northern Italy. The average heterozygosity was 0.68 for CAR/CAL polymorphism and 0.85 for RepIN20. The size of PCR fragments of CAR/CAL ranged from 207–225 bp and the most frequent allele was 207 bp (40.4%). The size of the fragments of RepIN20 ranged from 237–255 bp and the most frequent allele was 249 bp (30.8%). In the light of the highly polymorphic nature of both microsatellites and their intragenic location in SEL1L gene, we suggest that they could provide a means for linkage analysis to clarify the potential role of SEL1L in conferring susceptibility to IDDM or Grave's disease.     

枸骨IcFPS1基因的克隆、表达及生物信息学分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是三萜皂苷生物合途径的一个关键酶,为研究FPS基因在枸骨中的功能,该研究采用PCR技术将一个FPS基因的cDNA序列从枸骨叶中分离出来,并命名为IcFPS1。结果表明:根据测序结果分析发现扩增获得的IcFPS1基因cDNA长度为1 591 bp,包含一个完整的开放阅读框,大小为1 029 bp。通过序列分析发现枸骨IcFPS1基因编码342个氨基酸,分子量和等电点分别为39.58 kDa和5.18。通过理化性质预测分析发现IcFPS1蛋白不含信号肽,不含有跨膜区域,该IcFPS1蛋白为亲水性蛋白质。通过序列多重比对发现IcFPS1蛋白质与其他植物的FPS蛋白质高度同源,有共同的保守区域和氨基酸序列,其中与西洋参FPS序列的相似性高达89%。通过系统进化树分析发现枸骨FPS蛋白与同属于被子植物的五加科植物FPS蛋白亲缘关系较近,说明FPS基因在进化过程中相对比较保守。根据蛋白调控网络预测分析结果发现该蛋白可能与IPP1、IPP2、GGPS3、GGPS6和ERA1相互作用,参与类异戊二烯的合成代谢过程。通过实时荧光定量PCR分析发现IcFPS1基因在枸骨各个组织部位中均有表达,其中在枸骨根中表达量最高,在茎和雌花中表达量最低。     

赤桉ACT1和ACT2基因的克隆与生物信息学分析

为了解赤桉(Eucalyptus camaldulensis)肌动蛋白(Actin)在生长发育过程中的功能,根据赤桉幼苗转录组数据库中的肌动蛋白基因序列,从赤桉嫩叶中克隆了2条Actin基因片段,并利用RACE技术获得Actin基因的全长cDNA,分别命名为ECACT1和EC-ACT2基因。生物信息学分析表明,这两条基因的全长cDNA分别为1533 bp和1387 bp,均含有1个编码377个氨基酸的开放阅读框。经比对分析,赤桉Actin蛋白的氨基酸序列与其他植物Actin蛋白的具有较高的相似性,并且具有Actin蛋白特有的保守序列和相关特征。因此推测这两条基因对桉树的生长发育具有一定的调控作用。     

三浅裂野牵牛ItSGT1基因克隆及分析

SGT1(suppressor of the G2 allele of skpl)是多种植物抗病基因介导的抗病信号途径中的重要元件.该研究利用RT-PCR和RACE方法克隆出甘薯近缘野生种三浅裂野牵牛的SGT1基因,命名为ItSGT1.该基因含有一个长度为1 087 bp的开放阅读框,编码361个氨基酸,分子量约为40.1 kD,等电点为5.05.Blast及多序列比对分析表明,该基因与其他植物中的SGT1具有较高的相似性,且具有SGT1蛋白典型的功能域结构,即TPR区、VR1区、CS区、VR2区和SGS区.Southern杂交结果显示,SGT1基因在三浅裂野牵牛基因组中是多拷贝基因.组织特异性表达分析表明,ItSGT1基因在三浅裂野牵牛的根、茎和叶中均有表达.     

茶树CsCBF1基因克隆和转录活性分析

采用RACE技术克隆了一个受冷诱导的茶树CBF基因全长cDNA,命名为CsCBF1(GenBank登录号为EU563238)。CsCBF1cDNA全长序列为1 211bp,开放阅读框编码259个氨基酸。氨基酸序列分析表明,CsCBF1具有CBF家族典型的保守结构域,与其他植物的CBF具有较高的相似性;与拟南芥、辣椒和橡胶树编码的CBF相似性分别为56%、63%和56%。亚细胞定位结果表明,CsCBF1位于细胞核内。分别将10个CsCBF1缺失突变体与GAL4DNA结合域融合的结果显示,CsCBF1的羧基末端酸性结构域(第137位氨基酸至259位氨基酸)在酵母中具有转录激活活性。实时定量RT-PCR分析表明,CsCBF1基因受低温的快速诱导表达。     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

A cis-acting locus for the stable propagation of yeast plasmid pSR1

Summary A DNA plasmid resembling 2 m DNA of Saccharomyces cerevisiae, pSR1, isolated from a strain of Zygosaccharomyces rouxii, has a cis-acting region, Z, for plasmid stability. The Z region was delimited to a sequence of at most 383 bp in a small unique region of the plasmid. The Z region is high in A:T pairs and contains three different pairs of short (ca. 25 bp) inverted repeats with 65% to 79% homology and three copies of direct repeats of 24 to 27 bp in length with 67% to 72% homology, but does not encode a noteworthy open reading frame. It was suggested that the Z region interacts with the S product(s) encoded by the same plasmid and with a specific host factor, but not with the other stabilization factor encoded by the P locus on the sPR1 molecule.