水稻花药绒毡层特异表达基因RA39的克隆与表达特性分析

利用cDNA减法杂交,差异杂交筛选和RACE等技术。从水稻（Orza sativaL.ssp.japonica)中克隆了一个新的绒毡层特异性cDNA,其编码基因被命名为RA39。该cDNA长1013bp。编码由298个氨基酸残基组成的多肽RA39是一个单拷贝基因。在绒毡层细胞中特异性表达。在小孢子母细胞减数分裂期的绒毡层细胞中有较高的表达活性。用PSORT和PPSEARCH软件进行的结构分析揭示出RA39蛋白的N端是一个由17个氨基酸残基组成的信号肽,该蛋白包含一个跨膜区和一个胞质尾区两个主要结构域以及多个蛋白激酶的磷酸化位点。     

Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The mitochondrial elongation factor EF-Tu (tufM) in rice (Oryza sativa L.) was isolated and characterized. The rice tufM cDNA clone contained 1,726 nucleotides and coded for a 453 amino acid protein including a putative mitochondrial transit peptide of 64 amino acid residues. This coding region was composed of 12 exons and 11 introns. The deduced amino acid sequence showed 62% and 88% identities with rice chloroplast EF-Tu (tufA) and Arabidopsis mitochondrial EF-Tu, respectively. As previously observed for the rice tufA gene, the tufM gene is likely present as one copy in rice. The mitochondrial EF-Tu gene was differentially expressed during flower development, and the other translational EF-Tu genes (chloroplast EF-Tu and cytosolic EF-1 alpha) were also distinctly expressed in a temporal manner. Phylogenetic analysis of the rice tufM gene showed that the mitochondrial tufA homologue of Reclinomonas was more closely related to the mitochondrial tufM genes of flowering plants than fungal and other mitochondrial tuf genes. In addition, the tufM encoded an N-terminal extension showing significant similarity to that of rps14 (or sdhB), which is also a nuclear-encoded rice mitochondrial gene.     

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther’s tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species. Data deposition: The sequence reported in this paper have been deposited in the GeneBank database (Accession No. U12171)     

RT-PCR克隆甜菜硝酸还原酶cDNA全长序列及分析

根据GenBank中已公布的甜菜（Beta vulgaris）硝酸还原酶（nitrate reductase）基因序列（gb∣ABW05098.1∣）,设计引物,以50 mmol·L^-1 KNO₃溶液处理的甜菜幼苗为材料,从总RNA中通过RT-PCR分离得到一个硝酸还原酶基因,其cDNA长2 760 bp,包含了完整的基因编码序列,与已公布的硝酸还原酶基因序列相似性达99%。Southern杂交分析表明,硝酸还原酶基因在甜菜基因组中可能以两个拷贝或低拷贝形式存在。根据其编码的氨基酸序列,利用生物信息学预测了其亚细胞定位和蛋白质的三级结构。     

Purification and Characterization of Phospholipase D (PLD) from Rice (Oryza sativa L.) and Cloning of cDNA for PLD from Rice and Maize (Zea mays L.)

Phospholipase D (PLD) was purified to high homogeneity fromrice bran (Oryza sativa L.). Two peaks of PLD activity wereresolved by Mono Q anion-exchange chromatography. The molecularmass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDain gel filtration. Antibodies raised against the protein inone of the peaks precipitated the enzyme activities in bothpeaks. Enzymatic characteristics of PLD in the two peaks wereidentical except for a difference of 0.1 in the isoelectricpoints. Sequence analysis covering more than 10% of the aminoacids of the proteins and peptide mapping did not detect anydifference in the primary structure of the proteins. A cDNAfor PLD was isolated from rice and it encoded a protein of 812residues. The N-terminal sequences of purified PLDs matchedthe deduced amino acid sequence starting from residue 47. ANorthern blot showed this gene was expressed in leaves, roots,developing seeds and cultured cells, and a Southern blot detecteda single band of rice genomic DNA hybridizing to the cDNA. AcDNA for PLD was also isolated from maize. The similarity ofthe deduced amino acid sequences of PLD was 90% between riceand maize, 73% between the cereals and castor bean. ²Present address: Agribusiness Division, Japan Tobacco Inc.2-1,Toranomon, 2-chome, Minato-ku, Tokyo, 140 Japan     

Two rice <Emphasis Type="Italic">DMC1</Emphasis> genes are differentially expressed during meiosis and during haploid and diploid mitosis

We have cloned two rice homologues of yeast DMC1, a meiosis-specific gene required for recombination between homologous chromosomes. We show that rice DMC1A and DMC1B were produced by a gene duplication event that occurred after rice separated from the common ancestor of the cereals. The predicted proteins contain 344 amino acids, of which all but 7 are conserved between the two homologues. Between bases -1 and -245, the two promoters share six invariant blocks of sequence of 10-28 bp, interspersed in variable sequences. Both DMC1A and DMC1B are expressed in pollen mother cells coincident with meiosis, and in diploid non-meiotic tissues such as calli and root tips. DMC1B is also expressed in haploid male gametophytes during pollen maturation and in diploid zygotic embryos and endosperm after pollination. These data suggest that DMC1B, either alone or in combination with DMC1A, contributes to recombination during meiosis and during haploid and diploid mitosis.     

Lactate dehydrogenase from the Antarctic eelpout, Lycodichthys dearborni

As part of our studies to examine the molecular basis of cold-adaptation, we have determined the kinetic properties, thermal stability and deduced amino acid sequence of the enzyme lactate dehydrogenase (LDH) from an Antarctic zoarcid fish, Lycodichthys dearborni. Unlike Antarctic notothenioid fish which are endemic to the Southern Ocean, zoarcid fish are cosmopolitan and have a substantially longer evolutionary history as a sub-order. The A₄-LDH isoform was isolated and purified from the white muscle of L. dearborni. The kinetic parameters K_m^PYR and k_cat were determined at temperatures from 0 to 25°C. K_m^PYR was substantially higher at low temperatures than those from Antarctic and temperate notothenioid fish, whereas k_cat at these temperatures was essentially the same as those of the other fish LDH in this study. The sequence of L. dearborni A₄-LDH was determined from cDNA derived from white muscle RNA and found to be similar to, but distinct from, the A₄-LDH sequences of Antarctic notothenioid fish. Molecular modelling based on the structure of the A₄-LDH from Pagothenia borchgrevinki suggested that three conservative amino acid changes within the core of the protein that are not directly part of the active site but which might nonetheless influence the active site, may be important in cold-adaptation in L. dearborni A₄-LDH, and that several other changes on the surface of the protein might also play a role in cold-adaptation.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Molecular cloning,sequence and expression analysis of <Emphasis Type="Italic">ZmArf2</Emphasis>, a maize ADP-ribosylation factor

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

Molecular Cloning of a cDNA Encoding Ribosome Inactivating Protein from Amaranthus viridis and Its Expression in E. coli

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.