Chemical synthesis of a structure gene coding for small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco

A structural gene with 385 bp, which codes for the mature small subunit (rbcS) of ribulose-1,5-bi9phosphate carboxylase/oxygenase from tobacco, has been redesigned according to the codon usage of E.coli and chemically synthesized. To facilitate the systematic investigation of structure-functional relationship of the rbcS by site-specific mutagenesis, twenty one unique restriction sites distributed throughout the synthetic gene were designed. Gene synthesis was started from chemically synthesizing sixteen oligonucleotides each with 23-66 nucleotides, and these oligonu-cleotides were annealed to form eight duplexes from which 5'- and 3'- two half molecules were formed by stepwise T4 DNA ligase reaction, and then each half molecule was cloned into plasmid pWR13, after that, two half molecules were further confirmed by DNA sequencing, finally both half molecules excised from the cloning plasmid were recombinated to form plasmid pCOTrbcS containing the whole structural gene for tobacco rbcS.

IMMOBILIZATION AND SUBUNIT RECONSTITUTION OF RIBULOSE-1, 5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM RICE

Ribulose-1, 5-bisphosphate carboxylase/oxygenase ( EC 4.1.1.39 ) ( RuBP carboxylase ) is a bifunctional enzyme which catalyzes both carboxylation and oxygenation of ribulose-1, 5-bisphosphate (RuBP). These reactions are the first step in photosynthetic carbon fixation and photorespiration respectively. Because of its key role in the formation of plant yield, many studies on the structure of active-site and catalytic mechanism of this enzyme have

Transcritption regulation of soybean ribulose-1,5-bisphos-phate carboxylase small sub-unit gene by external factors

Ribulose-l,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4-8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and0.4% NaCl, respectively, is 2.5-3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accu-mulated with diurnal variation but easily influenced by light and low temperature.

Cloning and Sequence Analysis of rbcS Gene of Wild Barley (Hordeum brevisubulatum) under Salt Stress

[Objective] The aim was to study the cloning and sequence analysis of rbcS gene of wild barley under salt stress. [Method] The tender leaf blade of wild barley under salt stress was taken as the experimental material. The primers were designed according to the homology of rbcS gene sequences of wheat and barely in Genbank; then PCR amplification,recovery,ligation,transformation and sequencing of rbcS gene were carried out. [Result] Two rbcS genes including rbcS1 and rbcS2 with the length of 1 252 and 908 bp respectively were cloned from the barely genome. rbcS1 and rbcS2 were both composed by two exons and one intron. The exons length of the two genes was the same of 525 bp,encoding 174 amino acids,and the homology between them was 96%; however,the intron length of rbcS1 and rbcS2 was 448 and 107 bp respectively.

Pauciramus yunnanensis,gen.et sp.nov.,a novel freshwater red alga from China with proposal of the Ottiales ord.nov.(Nemaliophycidae,Rhodophyta)

Species of the red algal order Acrochaetiales mostly inhabit marine environments,with only two freshwater taxa Audouinella and Ottia.A new genus and species are described for freshwater red alga Pauciramus yunnanensis from Ailao Mountain,Yunnan,China.It is closely related to Ottia and a new order Ottiales was proposed for these genera.Pauciramus has unique combination of morphological characteristics including the following:plants caespitose and densely pulvinate,slender uniseriate filaments with well-developed rhizoids,rarely branched,cylindrical vegetative cell with a single,ribbon-shaped and parietal chloroplast,reproduction by tetrasporangia,and dense sporangial branchlet only at the upper portion of filaments.Phylogenetic analysis of sequence data from the plastid ribulose-1,5-bisphosphate carboxylase-oxygenase large-subunit(rbc L),small subunit gene of the ribosomal cistron(SSU)and barcode region near the 5′end of the mitochondrial cytochrome oxidase subunit I(COI-5P)indicated that:the new taxon,P.yunnanensis,was in a well-supported clade with Ottia meiospora,and this clade was sister to order Palmariales and Acrochaetiales.To adhere to the principle of monophyly,a new freshwater order Ottiales including Ottia and Pauciramus is proposed.Despite the high sequence interspecific divergences and obvious morphological differences between genera Ottia and Pauciramus,seems impractical to establish a new family for a monospecific genus.Therefore,we temporarily classified Pauciramus into the family Ottiaceae,and made necessary revisions to the description to accommodate this genus.

Adaptive Evolution of Large Subunits of RubisCO in Magnoliophyta Crops

[Objective] The aim was to study adaptive evolution of the large subunits of RubisCO in Magnoliophyta crops. [Method] Taking Magnoliophyta crops such as corn and rice as research materials, the analysis on molecular adaptive evolution was carded out by using codon replacement and maximum likelihood methods. [ Result] The RubisCO suffered positive selection effect and six amino acid sites were identified. [ Conclusion] The six amino acid sites are of important guiding significance for studying catalytic activity of RubisCO large subunits and crop improvement.

Isolation, Expression and Characterization of rbc L Gene from Ulva prolifera J. Agardh(Ulvophyceae, Chlorophyta)

Ulva prolifera is a typical green alga in subtidal areas and can grow tremendously fast. A highly efficient Rubisco enzyme which is encoded by Up Rbc L gene may contribute to the rapid growth. In this study, the full-length Up Rbc L open reading frame(ORF) was identified, which encoded a protein of 474 amino acids. Phylogenetic analysis of Up Rbc L sequences revealed that Chlorophyta had a closer genetic relationship with higher plants than with Rhodophyta and Phaeophyta. The two distinct residues(aa11 and aa91) were presumed to be unique for Rubisco catalytic activity. The predicted three-dimensional structure showed that one α/β-barrel existed in the C-terminal region, and the sites for Mg^(2+)coordination and CO_2 fixation were also located in this region. Gene expression profile indicated that Up Rbc L was expressed at a higher level under light exposure than in darkness. When the culture temperature reached 35℃, the expression level of Up Rbc L was 2.5-fold lower than at 15℃, and the carboxylase activity exhibited 13.8-fold decrease. Up Rbc L was heterologously expressed in E. coli and was purified by Ni^(2+) affinity chromatography. The physiological and biochemical characterization of recombinant Rubisco will be explored in the future.

Analysis of Short-Term Metabolic Alterations in Arabidopsis Following Changes in the Prevailing Environmental Conditions

Although a considerable increase in our knowledge concerning the importance of metabolic adjustments to unfavorable growth conditions has been recently provided, relatively little is known about the adjustments which occur in response to fluctuation in environmental factors. Evaluating the metabolic adjustments occurring under changing environmental conditions thus offers a good opportunity to increase our current understanding of the crosstalk between the major pathways which are affected by such conditions. To this end, plants growing under normal conditions were transferred to different light and temperature conditions which were anticipated to affect （amongst other processes） the rates of photosynthesis and photorespiration and characterized at the physiological, molecular, and metabolic levels following this transition. Our results revealed similar behavior in response to both treatments and imply a tight connec- tivity of photorespiration with the major pathways of plant metabolism. They further highlight that the majority of the regulation of these pathways is not mediated at the level of transcription but that leaf metabolism is rather pre-poised to adapt to changes in these input parameters.

Distribution characteristics and diversities of cbb and coxL genes in paddy soil profiles from southern China

The Calvin-Benson-Bassham cycle and Wood-Ljungdahl pathway are two well-known ones in the six carbon sequestration pathways,but the current knowledge of their occurrence in different layers of agricultural soil profiles is poor.In this study,the diversities of three genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubis CO),i.e.,genes encoding the green-like(cbbLG)and red-like(cbbLR)forms of Rubis COⅠ and encoding Rubis COⅡ(cbbM),and the gene encoding carbon monoxide dehydrogenase large subunit(coxL)from five paddy soils in southern China were investigated by real-time quantitative polymerase chain reaction,restriction fragment length polymorphism(RFLP)analysis,and clone library.The abundances of the four genes ranged from 10^(7) to 10^(9) copies g^(-1) soil,and the cbbLR gene outnumbered the other three genes in all soil samples,suggesting important roles they play in carbon dioxide(CO_(2))fixation.In addition,it was found that the copy numbers of cbbLR and cbbLG decreased with increasing soil depth,while the copy numbers of cbbM and coxL decreased in the shallow depths but increased with increasing soil depth.The results of RFLP showed a larger Shannon index(H)in the deeper soil layers among the four gene clone libraries,indicating that the community diversity in these soil layers was greater.The cbbLG gene had relatively low diversity(at genus level),and most of the sequences were classified as Sideroxydans and Thiobacillus.In contrast,the highly diverse groups were found in the other three gene clone libraries(cbbLR,cbbM,and coxL),most of which were distantly related to known sequences,even forming separate clusters.In summary,this study provides a new insight into CO_(2) fixers along agricultural soil profiles by comparing four bacterial genes.