Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Identification of terpene synthase gene family in Gynostemma pentaphyllum and expression pattern analysis under abiotic stresses / 中国中药杂志

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.

Chloroplast genomic characterization and phylogenetic analysis of Castanopsis hystrix / 生物工程学报

The structure and size of the chloroplast genome of Castanopsis hystrix was determined by Illumina HiSeq 2500 sequencing platform to understand the difference between C. hystrix and the chloroplast genome of the same genus, and the evolutionary position of C. hystrix in the genus, so as to facilitate species identification, genetic diversity analysis and resource conservation of the genus. Bioinformatics analysis was used to perform sequence assembly, annotation and characteristic analysis. R, Python, MISA, CodonW and MEGA 6 bioinformatics software were used to analyze the genome structure and number, codon bias, sequence repeats, simple sequence repeat (SSR) loci and phylogeny. The genome size of C. hystrix chloroplast was 153 754 bp, showing tetrad structure. A total of 130 genes were identified, including 85 coding genes, 37 tRNA genes and 8 rRNA genes. According to codon bias analysis, the average number of effective codons was 55.5, indicating that the codons were highly random and low in bias. Forty-five repeats and 111 SSR loci were detected by SSR and long repeat fragment analysis. Compared with the related species, chloroplast genome sequences were highly conserved, especially the protein coding sequences. Phylogenetic analysis showed that C. hystrix is closely related to the Hainanese cone. In summary, we obtained the basic information and phylogenetic position of the chloroplast genome of red cone, which will provide a preliminary basis for species identification, genetic diversity of natural populations and functional genomics research of C. hystrix.

Chloroplast genomic characterization and phylogenetic analysis of Pellionia scabra / 生物工程学报

Pellionia scabra belongs to the genus Pellionia in the family of Urticaceae, and is a high-quality wild vegetables with high nutritional value. In this study, high-throughput techniques were used to sequence, assemble and annotate the chloroplast genome. We also analyzed its structure, and construct the phylogenetic trees from the P. scabra to further study the chloroplast genome characteristics. The results showed that the chloroplast genome size was 153 220 bp, and the GC content was 36.4%, which belonged to the typical tetrad structure in P. scabra. The chloroplast genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes in P. scabra. Among them, 15 genes contained 1 intron, 2 genes contained 2 introns, and rps12 had trans-splicing, respectively. In P. scabra, chloroplast genomes could be divided into four categories, including 43 photosynthesis, 64 self-replication, other 7 coding proteins, and 4 unknown functions. A total of 51 073 codons were detected in the chloroplast genome, among which the codon encoding leucine (Leu) accounted for the largest proportion, and the codon preferred to use A and U bases. There were 72 simple sequence repeats (SSRs) in the chloroplast genome of P. scabra, containing 58 single nucleotides, 12 dinucleotides, 1 trinucleotide, and 1 tetranucleotide. The ycf1 gene expansion was present at the IRb/SSC boundary. The phylogenetic trees showed that P. scabra (OL800583) was most closely related to Elatostema stewardii (MZ292972), Elatostema dissectum (MK227819) and Elatostema laevissimum var. laevissimum (MN189961). Taken together, our results provide worthwhile information for understanding the identification, genetic evolution, and genomics research of P. scabra species.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey / Uma investigação sobre o cloroplasto e os genomas nucleares de táxons pertencentes ao subgênero Dracunculus (Bess.) Rydb. de Artemisia L. (Asteraceae) na Turquia

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and subspecific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn't been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Artemisia é um dos maiores gêneros da família Asteraceae, com cerca de 500 a 600 táxons em níveis específicos e subespecíficos e organizados em cinco subgêneros. Em razão do grande número de táxons, muitos taxonomistas estão tentando resolver o problema de sua classificação e filogenia, mas sua classificação natural ainda não foi alcançada. Nesta pesquisa, 60 indivíduos pertencentes a quatro táxons do subgênero Dracunculus de Artemisia L. na Turquia foram examinados. Para todos os indivíduos examinados de populações iguais e diferentes pertencentes aos táxons do subgênero Dracunculus, foram determinadas as sequências das regiões psbA-trnH do DNA do cloroplasto e ITS do DNA nuclear. Além disso, as regiões gênicas obtidas foram registradas no banco de dados do NCBI GenBank e um número de acesso foi obtido. Foi constatado que não houve fluxo gênico nem hibridização entre os quatro táxons estudados do subgênero Dracunculus, os quais também completaram sua especiação. De acordo com os resultados deste estudo molecular, A. campestris var. campestris, A. campestris var. marschalliana e A. campestris var. araratica foram propostos para ser elevados do nível de variedade para o nível de espécie. Esta pesquisa é importante porque é o primeiro estudo de base molecular relacionado com o subgênero Dracunculus em crescimento na Turquia.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Plastid transformation: advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Photosynthesis, growth, and survival in seedlings of four tropical fruit-tree species under intense radiation

In the Amazon region, agroforestry systems (AFSs) are recommended as a sustainable production alternative for local communities. A common component in Amazonian AFSs are tropical fruit trees, which can form the canopy or grow in the understory. In this study, we evaluated the effect of high radiation on photosynthesis, growth and seedling survival of four Amazonian fruit-tree species: Theobroma cacao, Eugenia stipitata, Inga edulis and Psidium guajava. Growth, chlorophyll fluorescence, gas exchange, and leaf pigments were measured in seedlings of each species grown for 12 months inside shade houses with low (8%), medium (30%) and high relative illumination (100%). Eugenia stipitata and T. cacao had the lowest acclimation capacity to high solar radiation, followed by I. edulis. Therefore, these species must be grown under intermediate light levels in early growth stages, to protect them from direct sunlight. In contrast, P. guajava seedlings demonstrated high tolerance to elevated radiation, therefore, this species can be planted under full sunlight. (AU)

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

In silico cloning and bioinformatics analysis of HSP21 in Manihot esculenta / 生物工程学报

HSP21 gene is a key gene to respond high temperature stress in plant and plays an important role in preventing protein denaturation, protecting cell structure and maintaining normal growth and development. Therefore, cloning HSP21 gene is the basis for revealing the molecular mechanism of resistance to high temperature stress in cassava. To obtain cassava HSP21 homologous gene and analyze the properties of predicted protein, electronic cloning technology was used to assemble and derivate new gene in this study, and bioinformatics analysis method was used to analyze the primary to highest structure, hydrophilicity/hydrophobicity, signal peptide, protein homology and phylogenetic evolution of expressed protein. HSP21 gene was 969 bp, its open reading frame was 705 bp, and the predicted protein contains 234 amino acids. The predicted protein is a non-transmembrane protein that is alkaline and hydrophilic, and is mainly localized in the chloroplast. Through multiple sequence alignment and phylogenetic analysis, it was found that the cassava HSP21 protein has high homology with other plants such as Hevea brasiliensis, Ricinus communis, and Jatropha curcas. The results could provide reference for the study of cloning and transformation of this gene.

Effects of light intensity on material and energy metabolism of Viola yedoensis / 中国中药杂志

Light energy is an important factor affecting plant growth. The hypothesis of "light-cold and heat property" holds that the original plants of traditional Chinese medicines(TCM) with cold property can obtain more energy to maintain growth in high light intensity environment, whereas the original plants of TCM with heat property prefer weak light environment. The current experiment investigated the effects of different light intensities on primary metabolites levels, energy levels, cell apoptosis, and leaves ultrastructure of Viola yedoensis, the original plants of TCM Violae Herba with cold property. There were five treatment groups of V. yedoensis, which was planted under Li1(8 500 lx),Li2(7 250 lx),Li3(6 000 lx),Li4(4 750 lx),Li5(3 500 lx)LEDs light intensity conditions, respectively. After harvest, primary metabolites levels, contents of ATP, ADP, AMP, activities of ATP synthesis and hydrolysis related enzyme, as well as cell apoptosis activation degree were measured, and transmission electron microscopy technique was used to observe leaves ultrastructure. The results showed that the total sugar, total protein, contents of ATP, ADP and AMP, activities of NADH dehydrogenase, cytochrome C reductase, ATP synthase and ATP hydrolase were positively correlated with light intensities(P<0.05). The crude fat content, activities of SDH and CCO enzyme showed a trend of increasing first and then decreasing, the highest value were found in Li2 group and Li3 group respectively(P<0.05). The vitality of caspase-3 and caspase-9 was negatively correlated with light intensities(P<0.05). The structure of chloroplast and mitochondria were normal and intact in Li1-Li3 groups, and the damage degree of Li4 and Li5 groups increased with the decrease of light intensities. The analysis of results indicated that the structure of chloroplast and mitochondria of V. yedoensis is normal under the light intensity of 6 000-8 500 lx, which can obtain more energy to maintain its growth and metabolism. When the light intensity is lower than 4 750 lx, the chloroplast morphology and mitochondrial membrane are damaged, affecting the metabolism of material and energy. There was no significant difference in energy charge of V. yedoensis in the light intensity range of 3 500～8 500 lx. The effect of light intensity on energy metabolism of V. yedoensis accords with the hypothesis of "light-cold and heat property".

Effects of light intensities on growth,physiological characteristic and chemical composition of Viola yedoensis / 中国中药杂志

The study is aimed to investigate the effects of light intensities on growth,photosynthetic physiology,antioxidant systems and chemical composition of Viola yedoensis and provide cultivation references for V.yedoensis.Five groups of V.yedoensis were planted under five light intensities conditions,namely 100%,80%,50%,35%,5%of full sunlight,and then morphological index,growth,chlorophyll fluorescence parameters,photosynthetic parameters and antioxidant enzyme system indexes were measured during harvest.The results showed that there was no significant difference in the biomass of V.yedoensis among 35% -100%full sunlight,but the biomass of those were significantly higher than that in the 5%full sunlight treatment(P<0.05).The net photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO_2 concentration and water use efficiency increased firstly and then decreased with the decrease of light intensity;F_m,F_v/F_mand Yield in 5% full sunlight treatment were significantly lower than those in the other four groups(P<0.05).The structure of chloroplast was normal under light intensity ranged from 50%to 100% full sunlight.The lamellar concentration of chloroplast matrix decreased and the starch granules decreased in 35% full sunlight treatment,and the margin of lamellar layer of chloroplast and substrate were blurred,and the starch granules were small and the number of starch granules decreased significantly under 5% full sunlight.MDA content in 5%full sunlight treatment was significantly higher than those in the other four groups(P<0.05).The total coumarin content and total flavonoid content decreased with the decrease of light intensity.In summary,the light in-tensity range suitable for the growth of V.yedoensis is wide(ranging from 35% to 100% full sunlight).The content of flavonoids and coumarins is positively correlated with light intensity.

Effects of changes in micro-weather conditions on structural features, total protein and carbohydrate content in leaves of the Atlantic rain forest tree golden trumpet (Tabebuia chrysotricha) / Efeito de alterações nas condições micro-climáticas sobre características estruturais, e proteína e carboidratos histoquimicamente marcados, de folhas da árvore da floresta Atlântica ipê amarelo

Abstract Golden trumpet, Tabebuia chrysotricha, is a native tree from the Brazilian Atlantic rain forest, with a broad latitudinal distribution. In this study, we investigated the potential effects of short-term changes in micro-weather conditions on structural features, and total protein and carbohydrate content of golden trumpet leaves, using structural and histochemical approaches. Leaves were harvested in four different micro-weather conditions: 1. Afternoon, after a hot, sunny day; 2. at dawn, after a previously hot, sunny day; 3. at noon, of a hot, sunny day; and 4. at noon, of a cold, cloudy day. Leaflets exposed to low light irradiance showed flattened chloroplasts, uniformly distributed within the cells, throughout the palisade parenchyma. Conversely, leaflets exposed to high light irradiance presented flattened and rounded chloroplasts, in the upper and lower palisade parenchyma cells, respectively. The strongest protein staining was found for leaves harvested at the coldest period, whereas the weakest protein staining was found for leaves harvested after a hot, sunny day. The largest and most numerous starch grains were found for leaves harvested in the afternoon, after a hot, sunny day. Conversely, the smallest and less numerous starch grains were found for leaves harvested at dawn. Analysis of the data reported herein suggests that the leaflet responses to transient changes in micro-weather conditions are likely to contribute to the golden trumpet successful establishment in the broad latitudinal distribution in which the species is found.

Resumo Ipê amarelo é uma árvore nativa da floresta Atlântica brasileira, encontrada em uma ampla distribuição latitudinal. Neste estudo, nós investigamos os efeitos potenciais de alterações de curto prazo nas condições micro-climáticas sobre características estruturais, proteína e carboidratos histoquimicamente marcados, de folhas de ipê amarelo, usando estratégias de análise estrutural e histoquímicas. As folhas foram marcadas em quatro condições microclimáticas distintas: 1. Tarde, após um dia quente e ensolarado; 2. Amanhecer, após um dia quente e ensolarado; 3. Ao meio-dia, de um dia quente e ensolarado; e 4. Ao meio-dia, de um dia frio e nublado. Folíolos expostos à baixa irradiância luminosa apresentaram cloroplastos achatados, uniformemente distribuídos no interior das células, por todo o parênquima paliçádico, enquanto que folíolos expostos à alta irradiância apresentaram cloroplastos achatados e arredondados, nas células superiores e inferiores do parênquima paliçádico, respectivamente. A marcação mais intensa para proteína foi observada para folhas coletadas no momento mais frio de coleta, enquanto que a marcação mais fraca foi observada para folhas coletadas após um dia quente e ensolarado. Os grãos de amido maiores e mais numerosos foram observados em folhas coletadas durante a tarde de dia quente e ensolarado, enquanto que os menores e menos numerosos grãos de amido foram observados em folhas coletadas ao amanhecer.

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

DNA barcoding identification of Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix based on trnL-trnF sequences / 中国中药杂志

To optimize indices of molecular identification for authentication of Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, four indices, including sequence similarity, specific positions, genetic distance and phylogenetic tree, were compared based on trnL-trnF sequences. Total DNA was extracted from Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, and trL-trnF sequences were amplified and sequenced. Sequence similarity was calculated by BLAST analysis. Specific positions were compared by DNAman software. Genetic distance and phylogenetic tree were analyzed by Mega software. The results showed that the inter-specific and intra-specific similarity of P. ginseng and P. quinquefolius respectively was 100% and 99. 6%. There were four specific positions at G153A, T463A, C732G and T818C. The inter-specific genetic distance (0) of trL-trnF sequences was lower than intra-specific genetic distance (0. 004). P. ginseng can be distinguished from P. quinquefolius based on the phylogenetic tree. It is concluded that Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix can be authenticated by identification indices of sequence similarity, specific positions, genetic distance and phylogenetic tree. Index of specific positions based on trnL-trnF sequences is the most efficient index to authenticate Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix.

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer / 药学学报

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.

Chloroplast ultra structure, photosynthesis and enzyme activities in regenerated plants of Stevia rebaudiana (Bert.) Bertoni as influenced by copper sulphate in the medium.

Stevia rebaudiana (Bert.) Bertoni is an important medicinal plant used as noncaloric commercial sweetener. Plants regenerated with higher levels of copper sulphate in the medium exhibited enhanced activity of peroxidase and polyphenoloxidase (PPO) enzymes. Transmission electron microscopy (TEM) revealed increase in size and number of electron dense inclusions in the chloroplasts of plants regenerated at optimised level of copper sulphate (0.5µM) in the medium. There was decrease in chlorogenic acid (CGA) content. Chl-a-fluorescence transient pattern (OJIP) showed that the photosynthesis process was more efficient at 0.5µM CuSO4 in the medium. 

High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts / 生物工程学报

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.