Carboxylase Large Subunit Research Articles

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters.

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.

First report of the Hawaiian genus Newhousia (Dictyotales, Phaeophyceae) from Madang, Papua New Guinea and description of the new species N. yhaga sp. nov.

Abstract The calcified encrusting brown-algal genus Newhousia was collected in 2012 from two sites in Madang Lagoon, northern Papua New Guinea. This is the first report of that enigmatic genus since its original description in 2004 from Oahu in the Hawaiian Islands. Morphologies of thalli from both Papuan localities agree with the original diagnosis and the published figures of the Hawaiian Newhousia imbricata. Ribulose-1,5-biphosphate carboxylase large subunit (rbcL) sequences of Papuan specimens were 96% identical to those of N. imbricata, and although there were no outstanding morphological differences between the Hawaiian and the Papuan specimens, the genetic divergence between the two strongly indicates they are not conspecific. A formal description of the new species, Newhousia yhaga, is provided and the phylogenetic implications of concatenated cox3, psbA and rbcL sequences evaluated. A multi-marker phylogeny was generated in the hope to establish the precise phylogenetic position of Newhousia within the order Dictyotales. Even though both Bayesian inference (BI) and maximum likelihood (ML) analysis point to Newhousia being sister to Lobophora, these results are not supported by ML bootstrap values. The present documentation of Newhousia in a southwest Pacific locality considerably extends the geographical range of this unusual genus.

Homologies between Bauhinia forficata Link subsp. pruinosa and pancreatic beta-cell specific transcriptional activator: a starting point for drug design new in diabetes?

Materials and methods Were performed the comparison between the AA sequence of the GenBank: CAA94019.1-ribulose 1,5biphosphate carboxylase large subunit, partial (chloroplast) [Bauhinia forficata subsp. pruinosa] and GenBank: BAC20389.1-pancreatic beta-cell specific transcriptional activator [Homo sapiens], available in the database of NCBI with the Basic Local Alignment Search Tool (BLASTp) software. Results The homology between the ribulose 1,5-biphosphate carboxylase large subunit, partial (chloroplast) [Bauhinia forficata subsp. pruinosa] and the pancreatic beta-cell specific transcriptional activator [Homo sapiens] ranged from 48.0% to 63.0% (Fig.1).

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants.

Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases.

In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.

Molecular distinction of two closely resembling Morinda species using rbcL and matK loci for quality management of Indian herbal medicines

Morinda reticulata Gamble and Morinda umbellata Linn. (Rubiaceae) are medicinally important climbers distributed as a mixed population in southern Western Ghats of India. A close morphological resemblance of these two species misleads the harvester in the identification of plant parts for preparation of herbal medicines. Though both species contain anthraquinone derivatives and share common medicinal properties for treating stomach disorders, each of these species has unique curative properties for treating selective diseases. Conventional methods are not reliable for identification of these species due to similarities in morphology. Thus, misidentification often leads to the deterioration of the quality of medicines. Thus, authentication utilizing conserved gene sequences in the chloroplast genome of these two Morinda spp. has been attempted for precise identification. Here we report the use of two barcoding genes (maturase kinase and ribulose 1,5-bisphosphate carboxylase large subunit) to distinguish M. reticulata and M. umbellata based on single nucleotide polymorphism. The present findings can be used for authenticating leaf samples of M. reticulata and M. umbellata.

Discrimination of Phellodendron amurense and P. chinense based on DNA analysis and the simultaneous analysis of alkaloids

Phellodendri Cortex is the bark of the stems of Phellodendron amurense Ruprecht or P. chinense Schneider (Rutaceae), which is orginated from periderm. The internal transcribed spacer sequences of 20 originated plants and identified samples were analyzed. The result showed that the 99% of the base sequences of P. amurense were identical to that of P. chinense, but the differentiation of P. amurense and P. chinense was difficult. In addition, the ribulose-1, 5-bisphospate carboxylase large subunit (rbcL) intergenic spacer sequences of specific parts produced the same result. However, when the analysis was carried out by using the RAPD (randomly amplification polymorphism DNA) analysis method, which utilizes 48 randomly primers, it allowed us to confirm the polymorphism of P. amurense and P. chinense in 12 primers. A high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of berberine, palmatine and jatrorrhizine in a traditional herbal drug, Phellodendri Cortex. The HPLC method was applied successfully to the quantification of three constituents in the extract of twenty Phellodendri Cortex. The results indicated that the established HPLC and RAPD methods are suitable for the quantitative analysis and the quality control multi-simultaneous discrimination in Phellodendri Cortex.

DNA identification of Salvia divinorum samples

Salvia divinorum (diviner's sage) is a plant in the mint family that produces an hallucinogenic compound, salvinorin A. The plant is used, often by chewing or smoking, as a "recreational" drug source and is regulated or banned in several states and countries. We describe a simple DNA technique, polymerase chain reaction of the ribulose bisphosphate carboxylase large subunit (rbcL) gene, that can distinguish S. divinorum leaf pieces from pieces of tobacco or cannabis. We have also found DNA sequences adjacent to the chloroplast leucine transfer RNA (trnL) gene that are specific to S. divinorum and distinguish it from other horticulturally popular Salvia species. We report some significant differences between the S. divinorum trnL sequences we determined and those now published in GenBank.

PCR-CE-SSCP used to authenticate edible oils

A useful procedure for the qualitative determination of edible vegetable oils adulteration has been developed. A CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism) method based on PCR (Polymerase-Chain-Reaction) technology was established to distinguish olive, soybean, sunflower, peanut, sesame, maize and corresponding edible oils, which are the most common edible oils in China. Two rbcL (ribulose 1,5-bisphosphate carboxylase large subunit) gene fragments were amplified from these 6 oil plants by 2 pairs of universal primers (uni4 and rbcl1) respectively and the amplicons were identified by CE-SSCP on ABI 3100 automatic sequence analyzer. Six oil plants were successfully differentiated by combined pattern of uni4 and rbcl1. Sensitivity of the method detecting DNA and oil mixture at different ratio was at level as low as 10% (V/V). The method developed was very suitable for the determination of modeled adulterants but it may also reveal an adulteration even if it does not derive from the adulterants employed in this study.

Survey of the Efficacy of a Short Fragment of the rbcL Gene as a Supplemental DNA Barcode for Diatoms

DNA barcoding is a tool that uses a short, standard segment of DNA to identify organisms. In diatoms, a consensus on an appropriate DNA barcode has not been reached, but several markers show promise. These include the 5.8S gene plus a fragment of the internal transcribed spacer 2 (ITS-2) of nuclear-encoded ribosomal RNA, a 420-bp segment of the 18S rRNA gene, and a 748-bp fragment at the 3'-end of the ribulose bisophosphate carboxylase large subunit (rbcL) gene. Here, we tested a 540-bp fragment 417-bp downstream of the start codon of the rbcL gene for its efficacy in distinguishing diatom species in a wide range of taxa. Overall, 381 sequences representing 66 genera and 245 species from the classes Mediophyceae and Bacillariophyceae were examined. Intra/interspecific thresholds were set at p=0.01 differences per site (diff./site) for Mediophyceae and p=0.02 diff./site for Bacillariophyceae and correctly segregated 96% and 93% of morphological congeners, respectively. When testing reproductively isolated or biological species, which are only available from Bacillariophyceae, 80% of species were discriminated. Therefore, we concluded that, alone, the rbcL region tested herein as potential a DNA barcode was not a sufficient discriminator of all diatoms. We suggest that this fragment could be used in a dual-locus barcode with the more variable 5.8S+ITS-2 to discriminate species without sufficient interspecific divergences in the tested rbcL region and to provide insight into species identity from a separately evolved genome.

Genotypic diversity and distribution of Ostreobium quekettii within scleractinian corals

The green filamentous endolithic alga Ostreobiumquekettii resides inside skeletons of scleractinian corals in close proximity with their tissue and plays a role in the viability of the coral and its associates. This study examined the distribution and diversity of O.quekettii within scleractinian corals from the Red Sea (Eilat, Gulf of Aqaba), using a molecular phylogenetic marker. The massive coral species Poriteslutea and Goniastreaperisi were sampled from a depth range of 6–55 m, and ribulose 1,5-bisphosphate carboxylase large subunit gene (rbcL) DNA sequence of the alga was amplified and analyzed for diversity and distribution of ecological patterns. This work reveals that O. quekettii has at least seven different clades distributed along a depth gradient in the examined scleractinian corals. Among the seven identified clades, four were found only in P. lutea, while the other two clades are found in both P. lutea and G. perisi. Goniastrea perisi colonies at depth of 30 m had a distinct O. quekettii clade that was absent in P. lutea. It is obvious from this study that the green endolithic alga O.quekettii is not a single genotype as previously considered but a complex of genotypes and that this differentiation is of ecological significance.

DNA Barcodes for Discriminating the Medicinal Plant Scutellaria baicalensis (Lamiaceae) and Its Adulterants

Scutellaria baicalensis GEORGI (Lamiaceae) is the botanical origin of the well-known traditional Chinese medicine "Huang Qin" (Radix Scutellariae). Due to overexploitation that had induced a decline in natural sources, the dried roots of its congeners, S. amoena, S. rehderiana, and S. viscidula, have been used to adulterate it in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, we sequenced and analyzed three candidate DNA barcodes, the ribosomal RNA maturase gene (matK), the ribulose-1,4-bisphosphate carboxylase large subunit gene (rbcL), and the psbA-trnH intergenic spacer (psbA-trnH), to discriminate S. baicalensis and its adulterants. All candidate DNA barcodes had been successfully amplified from leaf samples. Comparatively, only psbA-trnH had been yielded from commercially prepared crude drug samples. Based on the sequence divergence, rbcL can assign S. baicalensis and its adulterants into the correct family and genus, whereas, either matK or psbA-trnH can accurately discriminate S. baicalensis and its adulterants. We proposed the multilocus barcodes rbcL+psbA-trnH for the species identification of S. baicalensis and its adulterants, and the unique barcode psbA-trnH for the authentication of commercial Radix Scutellariae. The DNA barcoding technique could be applied to the quality control of "Huang Qin"-based medicinal preparations and to the management of medicinal herb trade in the markets.

English

The present study was undertaken to assess the role of plastid-rbc L (ribulose-1, 5-bisphosphate carboxylase large-subunit) gene sequences in addressing the evolutionary relationships within the angiosperms at inter and intra-familial levels using computational experiment. In order to elucidate the relationships, a set of 92 chloroplast rbc L sequences representing from 90 taxa of 12 genera and 10 angiospermic plant families (dicot and monocots) were withdrawn from the GenBank database. The multiple sequence alignment was performed using Genebee-ClustalW service to findout the regions of conserved or indels among the sequences. The phylogenetic tree was inferred from these sequences by employing Bootstrap method of UPGMA (Unweighted Pair Group Method with Arithmetic Mean) using MEGA (Molecular Evolutionary Genetics Analysis) software. The consistency of these generic-wise groupings was further confirmed by the MP (Maximum-Parsimony), ME (Minimum-Evolution) and NJ (Neighbor-Joining) methods. The analysis of these studies strongly indicates that, out of the 12 selected genera Trichosanthes (Cucurbitaceae) , Phyllanthus (Phyllanthaceae), Austrobryonia (Cucurbitaceae) , Solanum (Solanaceae) , Piper (Piperaceae) and Saxifraga (Saxifragaceae) are grouped into separate clusters and exhibiting monophyletic conditions. Where as, Drypetes (Putranjivaceae) , Asparagus ( Asparagaceae) , Cassia (Caesalpinioideae, sub-family) , Canna (Cannaceae) , Mentha (Lamiaceae) , are paraphyletic and the members of the Salvia (Lamiaceae) are distributed throughout these hiraeoid clades, confirming the polyphyly of this large genus. Similar observations were noticed in all four methods. Thus, chloroplast rbc L gene sequences can unambiguously resolve the relationships, as well as provided a good indication of major supra-generic groupings among the selected angiospermic plant families and also gave many clues for future studies.

Proteome Analysis of Resistance to Phytophora Root Rot in Soybean

The objective of this study was to survey and identify proteins in soybean ( Glycine max) cultivar Suinong 10 in response to inoculation of Phytophthora sojae by means of 2-dimensional gel electrophoresis and mass spectrometry techniques. In the 19 differentially expressed products obtained, 12 proteins were up-regulated and 6 proteins were down-regulated. The remaining one protein was specifically expressed after inoculation. Ten protein spots were assayed with biological mass spectrometry, including 1 specifically expressed, 1 down-regulated, and 8 up-regulated spots. These 10 proteins were involved in 8 functional proteins that were categorized into 4 groups: the ribolose-1,5-phosphate carboxylase large subunit and its precursor and succinyl coenzyme A comprising the metabolism group; kinase receptor like protein, oxidoreductase, and cysteamine ammonia acid oxidoreductase constituting the group of transport of metabolites in cells; the clothing zeta-3 protein subunits falling in the signal transduction group; and the F-box protein involved in jasmonic acid-mediated resistance. These differentially expressed proteins may be responsible for resistance of Suinong 10 to P. sojae and require further verifications.

Characterization of Cupric Glutamate Extinguishing Mechanism of Alexandrium sp. LC3 with Two-dimensional Electrophoresis and MALDI-TOF MS

Mechanisms by which cupric glutamate, a novel algicide, extinguishes Alexandrium sp. LC3 are shown in this study. We show that cupric glutamate not only stimulated the production of malonaldehyde (MDA) and dramatically promoted cell plasma membrane permeability (p < 0.01) but also remarkably reduced sulfhydryl (SH) group content (p < 0.01). Analysis of protein expression profiles by two-dimensional electrophoresis (2-DE) indicated that only 47 protein spots were detected in both control and cupric glutamate treated cells. Three reliable spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as ribulose-bisphosphate carboxylase large subunit precursor, RNA polymerase beta chain, and hypothetical protein, which can be well correlated with cupric glutamate stress. Based on above results, we hypothesize that the extinguishing mechanisms include (1) the cell membrane being damaged by cupric glutamate; (2) cupric glutamate probably induced denaturation and disintegration of intracellular protein, which led to inhibition of cell growth.

Phylogenetic relationships in Ephedra (Ephedraceae) inferred from chloroplast and nuclear DNA sequences

Sequences of the nuclear ribosomal DNA internal transcribed spacer region 1 and the chloroplast-encoded genes maturase K and ribulose-1,5 biphosphate carboxylase large subunit were obtained from species of Ephedra (Ephedraceae) representing the geographic range and morphological diversity of the genus. Phylogenetic analyses of the DNA data indicate that relationships within the genus are better predicted by geographic region of origin than by ovulate cone characters. The sampled species with dry, winged (versus fleshy) ovulate cone bracts or single-seeded cones do not form monophyletic groups and therefore the previous classification systems of Ephedra based on these aspects of bract morphology appear to be largely unnatural. Three groups were identified among the Old World species studied, one comprising European and Mediterranean species and two including only Asian species. The sequence data suggest a possible early divergence of a New World clade of Ephedra from among the Old World groups. The South American species form a distinct clade apparently related to one of two groups of North American species, which accords with a frequent floristic pattern of close relationships between species groups in western South America and southwestern North America.

Performing microchannel temperature cycling reactions using reciprocating reagent shuttling along a radial temperature gradient

This study develops a novel temperature cycling strategy for executing temperature cycling reactions in laser-etched poly(methylmethacrylate) (PMMA) microfluidic chips. The developed microfluidic chip is circular in shape and is clamped in contact with a circular ITO heater chip of an equivalent diameter. Both chips are fabricated using an economic and versatile laser scribing process. Using this arrangement, a self-sustained radial temperature gradient is generated within the microfluidic chip without the need to thermally isolate the different temperature zones. This study demonstrates the temperature cycling capabilities of the reported microfluidic device by a polymerase chain reaction (PCR) process using ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) gene as a template. The temperature ramping rate of the sample inside the microchannel is determined from the spectral change of a thermochromic liquid crystal (TLC) solution pumped into the channel. The present results confirm that a rapid thermal cycling effect is achieved despite the low thermal conductivity of the PMMA substrate. Using IR thermometry, it is found that the radial temperature gradient of the chip is approximately 2 degrees C mm(-1). The simple system presented in this study has considerable potential for miniaturizing complex integrated reactions requiring different cycling parameters.

AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes

To increase the contents of specific free amino acids in the rice ( Oryza sativa L.) cultivar “Donganbyeo”, mutant cell lines resistant to growth inhibition by S-(2-aminoethyl)-cysteine (AEC) were selected from calli irradiated with gamma-rays through embryo culture. In the four AEC resistant M 3 lines, LR3–69, LR5–132, LR7–2 and LR9–48, the amino acids contents were 26, 7, 29 and 37%, respectively, greater than in the “Donganbyeo”. The most significant amino acid increases in the resistant lines were in lysine and tryptophan levels. The lysine levels of the four resistant lines were 2.66, 1.58, 2.37 and 2.29 times, higher than in the “Donganbyeo”. Total proteins extracted and resolved by SDS–PAGE, showed few differences in the protein profiles of the control and mutant lines. In glutelins, SDS–PAGE protein patterns showed two typical mutant protein bands at 24 kDa. The SDS–PAGE profiles of mutant prolamines revealed two distinct bands of different intensities, at ca. 22 and 18 kDa. AEC may inhibit cell growth irreversibly by competing with lysine for incorporation into protein. This incorporation presumably leads to an altered protein conformation and function, and accounts for the cytotoxic effects of AEC. Therefore, AEC creates stresses in vivo much like other environmental stresses. Changes in gene expression in response to AEC were analyzed by 2-dimensional gel electrophoresis (2-DE). The number of new protein spots found in resistant mutants of LR3–69, LR5–132 and LR9–48 were 4, 11 and 16, respectively. De novo synthesized and enhanced proteins in these mutant lines after induction with AEC were 13, 11 and 9, and 7, 12 and 13 spots, respectively. The five proteins (L1, L2, L5, L6 and L7) were identified as a 33 kDa oxygen-evolving protein of photosystem II, ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit, ascorbate peroxidase (APX), glutamine synthetase (GS) and Cu/Zn superoxide dismutase (Cu/Zn SOD). Based on the 2-DE results, the AEC stress-mediated responses of two antioxidant enzymes, SOD and APX, were examined. Compared with a 50.3% increase in the control, increases in the rates of SOD activity of LR3–69, LR7–2 and LR9–48 were 136.5, 177.1 and 184.8%, respectively. After AEC treatment, the APX activity level in the control increased by 5.4%, whereas those of the mutant lines, LR3–69, LR5–132, LR7–2 and LR9–48, increased to 1.6, 1.4, 2.4 and 4.0 times that of the untreated control, respectively.

AEC resistant rice mutants induced by gamma-ray irradiation may include both elevated lysine production and increased activity of stress related enzymes

To increase the contents of specific free amino acids in the rice (Oryza sativa L.) cultivar “Donganbyeo”, mutant cell lines resistant to growth inhibition by S-(2-aminoethyl)-cysteine (AEC) were selected from calli irradiated with gamma-rays through embryo culture. In the four AEC resistant M3 lines, LR3–69, LR5–132, LR7–2 and LR9–48, the amino acids contents were 26, 7, 29 and 37%, respectively, greater than in the “Donganbyeo”. The most significant amino acid increases in the resistant lines were in lysine and tryptophan levels. The lysine levels of the four resistant lines were 2.66, 1.58, 2.37 and 2.29 times, higher than in the “Donganbyeo”. Total proteins extracted and resolved by SDS–PAGE, showed few differences in the protein profiles of the control and mutant lines. In glutelins, SDS–PAGE protein patterns showed two typical mutant protein bands at 24 kDa. The SDS–PAGE profiles of mutant prolamines revealed two distinct bands of different intensities, at ca. 22 and 18 kDa. AEC may inhibit cell growth irreversibly by competing with lysine for incorporation into protein. This incorporation presumably leads to an altered protein conformation and function, and accounts for the cytotoxic effects of AEC. Therefore, AEC creates stresses in vivo much like other environmental stresses. Changes in gene expression in response to AEC were analyzed by 2-dimensional gel electrophoresis (2-DE). The number of new protein spots found in resistant mutants of LR3–69, LR5–132 and LR9–48 were 4, 11 and 16, respectively. De novo synthesized and enhanced proteins in these mutant lines after induction with AEC were 13, 11 and 9, and 7, 12 and 13 spots, respectively. The five proteins (L1, L2, L5, L6 and L7) were identified as a 33 kDa oxygen-evolving protein of photosystem II, ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit, ascorbate peroxidase (APX), glutamine synthetase (GS) and Cu/Zn superoxide dismutase (Cu/Zn SOD). Based on the 2-DE results, the AEC stress-mediated responses of two antioxidant enzymes, SOD and APX, were examined. Compared with a 50.3% increase in the control, increases in the rates of SOD activity of LR3–69, LR7–2 and LR9–48 were 136.5, 177.1 and 184.8%, respectively. After AEC treatment, the APX activity level in the control increased by 5.4%, whereas those of the mutant lines, LR3–69, LR5–132, LR7–2 and LR9–48, increased to 1.6, 1.4, 2.4 and 4.0 times that of the untreated control, respectively.

Maternal inheritance of chloroplast DNA in intergeneric sexual hybrids of “true citrus fruit trees” revealed by PCR-RFLP analysis

SummaryPCR-RFLP analysis was employed to clarify the inheritance pattern of chloroplast DNA (cpDNA) in six kinds of intergeneric sexual hybrids among five genera, i.e. Fortunella, Eremocitrus, Poncirus, Microcitrus and Citrus within the “true citrus fruit trees” group of the subtribe Citrinae (Rutaceae). Two cpDNA gene regions were amplified from each material via PCR as following: one region of tRNA (trnD-trnT) and a variable region bounded by the conserved sequences in ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) and open reading frame 106 (ORF106). Twelve restriction endonucleases (MspI, XbaI, RsaI, AluI, HinfI, ScrFI, StyI, HaeIII, Sau3AI, NdeII, HhaI and TaqI) were used for RFLP analysis of the amplified regions of cpDNA. Polymorphisms were detected with five (HinfI, RsaI, NdeII, ScrFI and XbaI) and four (HinfI, RsaI, HaeIII and Sau3AI) enzymes for rbcL-ORFl06 and trnD-trnT regions, respectively. All progeny examined in each kind of the intergeneric sexual hybrid exhibited maternal inheritance of cpDNA. Also, cpDNA analysis for determining the maternal parent of a natural hybrid, Microcitrus virgata (Sydney hybrid) indicated that M. australis (Planch.) Swing. was the female parent of M. virgata.