Aims: Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447). Methodology and results: Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli. Conclusion, significance and impact of study GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.
Synechococcus ; Ribulose-Bisphosphate Carboxylase ; Escherichia coli ; Holoenzymes

The aim of this study was to establish a PCR for detecting of the hepatitis B virus (HBV) in blood and blood products. A primer pair set was designed to amplify a 513 bp fragment in the S-region of the HBV genome in the first PCR and a 233 bp fragment of first PCR amplicon in the second PCR with Rubisco (internal control). In order to assess the specificity of the PCR results, all the samples were tested cross-reactivity or interference in the assay. This method did not result in cross-reactivity with the non-HBV (HAV, HCV, HIV, CMV, HPV 18&6b, parvovirus B19/ or HSV 1&2) positive samples and was unaffected. In case of the HBV spiked blood products such as the immunogloubulin and coagulation factors, the lower detection limit of this method for the HBV DNA is 62.5 IU/ml. The PCR method is fully established in this study and will be a valuable method for the detection of the HBV in a variety of blood products, particularly, those derived from starting materials with a high titer of virus.
Blood Coagulation Factors ; DNA ; Genome ; Hepatitis B virus ; HIV ; Limit of Detection ; Parvovirus ; Polymerase Chain Reaction* ; Ribulose-Bisphosphate Carboxylase ; Sensitivity and Specificity

OBJECTIVEPoisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China.

METHODSSeventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes (matK, rbcL, and ITS) were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment (PWG) distance, and Tree-Building were tested for discrimination power.

RESULTSThe primer universality of all the three markers was high. Except in the case of ITS for Hemerocallis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method.

CONCLUSIONDNA barcoding technique provides us a fast tool for clinical identification of poisonous plants in China. We suggest matK, rbcL, ITS used in combination as DNA barcodes for authentication of poisonous plants.

China ; DNA Barcoding, Taxonomic ; standards ; DNA Primers ; genetics ; DNA, Intergenic ; genetics ; Plant Proteins ; genetics ; Plants, Toxic ; classification ; genetics ; Ribulose-Bisphosphate Carboxylase ; genetics ; Sequence Analysis, DNA ; Species Specificity

Photosynthetic CO(2) fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO(2) fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO(2). The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.
Amino Acid Substitution ; Bacterial Proteins ; chemistry ; genetics ; Carbon Dioxide ; chemistry ; Directed Molecular Evolution ; Escherichia coli ; growth & development ; Ribulose-Bisphosphate Carboxylase ; chemistry ; genetics ; Synechococcus ; enzymology

AIMTo identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis.

METHODSGeneral DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer.

RESULTSA fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately.

CONCLUSIONThe sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.

Base Sequence ; Chloroplasts ; genetics ; DNA, Plant ; analysis ; Genes, Plant ; Iridaceae ; classification ; genetics ; Iris Plant ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Ribulose-Bisphosphate Carboxylase ; classification ; genetics ; Sequence Analysis, DNA ; Species Specificity

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.
Base Sequence ; Chloroplasts ; genetics ; Cluster Analysis ; DNA Barcoding, Taxonomic ; DNA, Chloroplast ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Drugs, Chinese Herbal ; chemistry ; Forsythia ; chemistry ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; Ribulose-Bisphosphate Carboxylase ; genetics ; Sequence Analysis, DNA ; Species Specificity