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…     

Cloning and Structure Analysis of rbcS Gene from Wild Soybean

About 1 kb fragment of rbcS (ribulose 1, 5-bisphosphate carboxylase small subunit) gene in wild soybean (Glycine soja, Ji 50017) was amplified from total DNA by PCR assay. Sequence analysis of the fragment indicated that 1089 bp sequenced included the whole coding region for Rubisco small snbunit. The rbcS gene in wild soybean encoded a precursor composed of a transit peptide of 55 amino acids and a mature protein of 123 amino acids. There were two introns found in the rbcS gene as other dicotyledonous species previously sequenced. Comparison of DNA sequences showed high degree homology of rbcS genes between wild soybean and cultivated soybean (Glycine max var. wayne). Some changes of amino acids emerged from the diverse nucleotides did not affect the function of the small subunit. These results may contribute some basic data in molecular biology to study the origin and evolution of soybean.     

Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) located in the chloroplast is the most abundantprotein in the leaves of light-grown plants. This enzymecatalyzes the first step in net photosynthetic CO2 fixationand photorespiration. The native Rub…     

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.     

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.     

Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.     

A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor.

Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301. The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E. coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes. Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively. Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP. The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate. Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO. Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins. These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.     

Gene phylogenies and the endosymbiotic origin of plastids.

The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.     

Sequences 5'' to translation start regulate expression of petunia rbcS genes.

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes.     

Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.

Expression of rbcS genes encoding small subunit of rubisco, most abundant protein in green tissue, is regulated by at least three parameters--tissue type, light conditions and stage of development. One of the green tissue-specific promoters of rbcS gene family was isolated from pigeonpea by PCR. Expression of uidA gene encoding beta-glucuronidase in the transgenic tobacco plants under the control of pigeonpea rbcS promoter, clearly showed that this promoter was as strong as pea rbcS3A promoter characterized earlier. Study of the sequence similarity with pea rbcS3A promoter, especially the region (boxes I and III) that is required for rbcS3A expression, showed more than 50% divergence. In contrast, pigeonpea promoter sequence isolated in the present study was more similar to that of spinach and rice rbcS promoters.     

Overlapping reading frames in Oenothera mitochondria

An open reading frame (ORF) preceding the cytochrome oxidase subunit II (CO II) gene in Oenothera mitochondria has four nucleotides in common with this gene. The last two nucleotides of the CO II initiation codon ATG are the first two nucleotides of the TGA termination codon in the upstream ORF. Both reading frames are cotranscribed in a bicistronic mRNA species of 1250 nucleotides in length in Oenothera. The open reading frame codes for a protein of 58 amino acids with structural homology to the ATPase subunit 8 genes in fungal and mammalian mitochondria. Using coding space optimally though overlapping genes appears to be without economical reason considering the large size of higher plant mitochondrial genomes.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。