野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…     

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1.

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.     

Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes.

Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB. rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits. rbcL-rbcS was the same as that reported previously (A. M. Viale, H. Kobayashi, T. Takabe, and T. Akazawa, FEBS Lett. 192:283-288, 1985). A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced. We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites. rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively. Edman degradation analysis of the subunits of RuBisCO isolated from C. vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium. The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively. Among hetero-oligomeric RuBisCOs, the C. vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%). A similar situation has been observed for the C. vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits.     

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin)

Abstract The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fim A, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.     

叶绿体超氧化物歧化酶（ChlSOD）基因的构建及序列分析

构建叶绿体超氧化物歧化酶基因（ChlSOD),采用RT-PCR方法分离豌豆RUBP羧化酶小亚基导肽基因（TP）,定向克隆至pUC19测序,定向克隆烟草MnSOD成熟蛋白基因（SODm)至pUC19;采用平粘端连接法将二者在pUC19中构成嵌合基因ChlSOD,并对此基因进行序列分析,序列分析表明：TPcDNATP,12bp的Linker及615bp SODm。TP与ChlSOD基因的序列分析与国外报道序列完全吻合。     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

Cloning, sequence, and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene

Genomic DNA containing the protein coding region for Drosophila cAMP-dependent protein kinase catalytic subunit has been cloned and sequenced. The probe used to detect and isolate the gene fragment was constructed from two partially complementary synthetic oligonucleotides and contains 60 base pairs that encode (using Drosophila codon preferences) amino acids 195-214 of the beef heart catalytic subunit. In reduced stringency hybridization conditions, the probe recognizes two target sites in fly genomic DNA with 85% homology. One of these sites is in the cAMP-dependent protein kinase catalytic subunit gene, which was isolated as a 3959-base pair HindIII fragment. This fragment contains all of the protein coding portion, 900 base pairs upstream of the initiator ATG, and 2000 base pairs downstream of the termination codon (TAG). The coding portion of the gene contains no introns and yields a protein of 352 amino acids. There is a 2-amino acid insertion near the N terminus of the fly protein relative to the beef and mouse enzymes. Of the remaining 350 amino acids, 273 are invariant in the three species. A probe derived from the coding sequence of the HindIII clone hybridizes strongly to a 5100-base poly(A)+ RNA and weakly to 4100- and 3400-base poly(A)+ RNAs expressed in adult flies. A 2100-base pair EcoRI genomic fragment containing the second site recognized by the 60-base pair probe has also been cloned. DNA sequence analysis demonstrates that this fragment is part of the cGMP-dependent protein kinase gene or a close homolog. The catalytic subunit gene and the cGMP-dependent protein kinase gene have been located in regions 30C and 21D, respectively, of chromosome 2.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.     

Nucleotide sequence of yeast gene CP A1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Homology of the deduced amino acid sequence to other glutamine amidotransferases

A yeast DNA fragment carrying the gene CP A1 encoding the small subunit of the arginine pathway carbamoyl-phosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CP A1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45 358 and showing extensive homology with the product of carA, the homologous Escherichia coli gene. CP A1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl-phosphate. A comparison of the amino acid sequences of CP A1 polypeptide with the glutamine amidotransferase domains of anthranilate and p-amino-benzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11-residue oligopeptide contains a cysteine which is considered as the active-site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia-dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process. The flanking regions of gene CP A1 have been sequenced, 803 base pairs being determined on the 5' side and 382 on the 3' side. Several features of the 5'-upstream region of CP A1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T-G-A-C-T-C) previously identified in several genes subject to the general control of amino acid biosynthesis.     

野生大豆P5CS基因的克隆及对盐胁迫反应

逆境下植物大量积累脯氨酸是减轻胁迫伤害的一种自我保护机制。本研究应用同源克隆方法从NaCl处理的野生大豆中克隆获得一个脯氨酸合成酶(P5CS)基因,命名为GsP5CS。该基因核苷酸序列全长2.232 kb,含一个2148bp开放阅读框,编码715个氨基酸,包含有高等植物P5CS蛋白质的5个主要功能域,与菜豆PvP5CS1基因核苷酸序列相似性高达98.79%。Real Time PCR分析显示该基因受轻度盐胁迫诱导上调表达,根中表达高峰出现在200 mmol/L NaCl处理下,相对表达量为对照的5.83倍;叶片中表达高峰出现在300 mmol/L NaCl处理条件下,相对表达量为对照的12.78倍。并且该基因在根和叶片中的表达模式和脯氨酸含量的变化模式相同。上述结果说明,GsP5CS可能参与野生大豆脯氨酸合成。     

Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis

An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.     

Organization of the 5S rRNA genes in the soybean Glycine max (L.) Merrill and conservation of the 5S rDNA repeat structure in higher plants

The 5S rRNA gene of the soybean Glycine max (L.) Merr. has been cloned on a 556-bp fragment of DNA and sequenced. This fragment contains two copies of the soybean 5S rDNA sequence, one intact and one truncated, separated by noncoding DNA. We have used this clone to investigate the organization of the 5S genes within the soybean genome and the extent of their methylation. Our results demonstrate that soybean 5S genes are clustered, organized into tandem repeats of 330 bp, and extensively methylated. Hybridization of the 5S sequence to Southern transfers of soybean DNA digested with BamHI reveals a striking ladderlike pattern. Hybridization of the soybean 5S sequence to a wide variety of plant DNAs results in similar patterns, suggesting that the 5S rDNA sequence, gene organization, and methylation pattern are conserved in many higher plants.     

Cloning and sequencing of the structural gene for the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1: evidence for two tryptophan residues involved in the active center

In two independent clone libraries, clones were identified that hybridized with oligonucleotide probes based on N- or C-terminal polypeptide sequence of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1. Plasmids from all clones had in common a 5.2 kb Bam HI-HindIII DNA fragment. A 0.57 kb SacII-BclI subfragment that hybridized to the oligonucleotide probes was sequenced. Nucleotide sequence analysis coincided with polypeptide sequence data in the structural part of the small subunit with a single contradiction: amino acid 17 is Asp rather than Asn. The two amino acids that are involved in the active center which had not been determined from previous polypeptide sequencing proved to be tryptophans.     

大豆异黄酮合酶基因的克隆及序列分析

目的:大豆异黄酮是多酚类混合物,有防治肿瘤发生,提高机体免疫力等多种保健功能。异黄酮合酶(isoflavone synthase,IFS)是合成异黄酮的关键酶。本文为了利用异黄酮的特有生物学功能,从大豆中克隆了该基因。方法:采用PCR扩增从大豆[Glycine max(Linn.)Merr.]总RNA中分离了异黄酮合酶基因,并将其克隆到pUCm-T载体并测序。结果:得到全长1583bp的片段。以期用于构建诱导表达基因敲除系统,并用于无性繁殖植物的无标记基因转化。结论:序列分析表明,异黄酮合酶基因(IFS1)含1583个核苷酸,与已报道的序列比较,核苷酸的同源性为92%。     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

Nucleotide sequence of the Saccharomyces cerevisiae CTT1 gene and deduced amino-acid sequence of yeast catalase T

A 2642-base-pair DNA fragment containing the catalase T (CTT1) structural gene of the yeast Saccharomyces cerevisiae and its flanking regions has been sequenced. The gene codes for a protein of 562 amino acids (relative molecular mass 64,449) and appears to contain no intron. The amino acid sequence of catalase T derived from the DNA sequence shows 40.7% homology (52.2% including conservative replacements) to that of bovine liver catalase. All amino acids previously postulated to participate directly in catalysis by liver catalase and most of the amino acids of the immediate environment of hemin, the prosthetic group of catalase, are conserved in catalase T. The data obtained indicate that the folding of polypeptide chains of the two catalases compared has been conserved within a central region consisting mainly of the beta-barrel domain, which bears the prosthetic group, and a major part of the "wrapping domain". N- and C-terminal regions involved in subunit interactions are less well conserved. It is suggested that their structure is more similar to that of the corresponding regions of Penicillium vitale catalase. However, catalase T lacks the C-terminal flavodoxin-like domain present in this protein.