Cloning and gene functional analysis study of dynamin-related protein GeDRP1E gene in Gastrodia elata / 药学学报

The gene GeDRP1E encoding dynamin-related protein 1E in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDRP1E gene was carried out by using ExPASy, ClustalW, MEGA, etc. Positive transgenic Arabidopsis plant and potato minituber were obtained with the genetic transformation system of Arabidopsis and potato. The plant height and seed setting rate of transgenic Arabidopsis, and agronomic characters, such as size, weight and starch content of potato minituber of transgenic potato were tested and analyzed. And GeDRP1E gene function was preliminarily investigated. The results showed that the open reading frame of GeDRP1E gene was 1 899 bp in length and 632 amino acids residues were encoded, with a relative molecular weight of 69.90 kDa and a molecular formula of C3079H4973N883O933S19. It was predicted that the theoretical isoelectric point was 7.27, the instability coefficient was 43.34, and the average hydrophilicity index was -0.259, which was indicative of an unstable hydrophilic protein. GeDRP1E has no transmembrane structure and signal peptide, and was localized in the cytoplasm. The phylogenetic tree showed that GeDRP1E was highly homologous with DRP1E proteins of other plant species, among which GeDRP1E had the highest homology with DcDRP1E (XP_020689662.1) in Dendrobium candidum, reaching 90.05%. GeDRP1E plant expression vector pCambia1300-35Spro-GeDRP1E was constructed by double digests, and Arabidopsis complementary mutant and potato overexpression strain of GeDRP1E gene were obtained by Agrobacterium-mediated gene transformation. Compared with the Arabidopsis AtDRP1E mutant, the height and seed setting rate of the GeDRP1E complementation mutant were rescued. The minituber of GeDRP1E overexpression potato had larger size, heavier weight and higher starch content, comparing to wild-type potato. It was preliminarily induced that GeDRP1E was involved in mitochondrial morphology regulation, which related to the growth and development of Arabidopsis plants and potato miniature. The research results laid a foundation for further elucidating the molecular mechanisms underlying the growth and development of G. elata tuber development.

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata / 中国中药杂志

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Soybean GmGolS2-2 improves drought resistance of transgenic tobacco / 生物工程学报

Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

Cloning and structural elucidation of a brassinosteroids biosynthetic gene (Atdwarf4) and genetic transformation of Indian mustard (Brassica juncea L.)

Phytohormones play critical roles in plant growth and development. Brassinosteroids (BRs) are essential group of phytohormones required for optimum growth of plants and their deficiency causes distinctive dwarf phenotypes in plants. Homeostasis of BRs in plants is maintained by DWARF4 enzyme that mediates multiple 22?-hydroxylation steps in brassinosteroid biosynthesis. Arabidopsis plants over-expressing DWARF4 show increase in inflorescence, number of branches and siliques; thereby increased number of seeds/plant. This suggests that engineering DWARF4 biosynthesis in Brassica plant can be strategized to enhance yield in mustard. In the present study (i) we cloned dwarf4 gene from Arabidopsis using gene specific PCR strategy, (ii) elucidated the three-dimensional structure of DWARF4 protein at molecular level where it revealed presence of four beta sheets and 20 alpha-helices, and (iii) transformed mustard cultivar Pusa Jaikisan with an objective to develop transgenic mustard with enhanced number of siliques. We obtained several putative transgenics with an average transformation efficiency of 3.3%. Molecular characterization with nptII specific primers confirmed presence of transgene in six putative transgenic plants.

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Optimization of Agrobacterium-Mediated Genetic Transformation System of Armillaria gallica / 中国实验方剂学杂志

ObjectiveTo optimize the existing genetic transformation system of Armillaria gallica to improve the transformation efficiency and lay a foundation for the follow-up research on Armillaria molecular marker-assisted breeding and gene function. MethodThe genetically transformed plasmid pH101-PAgGPD-GFP-TrpC was constructed，transformed into Escherichia coli，amplified， and cultured，and the plasmid was extracted. The extracted plasmid was transformed into four different agrobacteria LBA4404，EHA105，GV3101，and AGL-1，respectively. The transformed agrobacteria were used for impregnating A. gallica，and the agrobacteria with the highest conversion rate were screened out. Then the agrobacterium-mediated genetic transformation system of A. gallica was optimized from the type and concentration of antibiotics，co-culture time，concentration of bacterial solution， and impregnation method. The phenotype profiles of A. gallica under different conditions were observed using Synbiosis ProtoCol 3. ResultThe optimized genetic transformation conditions of A. gallica were as follows： the Agrobacterium strain of EHA105 at absorbance A600 nm=0.6， the co-culture time of 2 d， the infection mode of negative pressure impregnation for 10 min， the primary screening medium of PDA medium containing 400 mg·L-1 cefotaxime sodium and 10 mg·L-1 hygromycin，and the secondary screening medium of PDA medium containing 12 mg·L-1 hygromycin. ConclusionIn this study，the existing genetic transformation system of A. gallica was optimized，and there was a significant difference in the transformation rate before and after optimization （P<0.05）. After optimization，the transformation efficiency of A. gallica was about 4.33%，which was about eight times higher than that before optimization.

Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175 / 口腔疾病防治

Objective@#To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function.@*Method @# Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated.@* Results @# The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001).@* Conclusion@# The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.

High-throughput sequencing reveals the molecular mechanisms determining the stay-green characteristic in soybeans

Senescence is an internally systematized degeneration process leading to death in plants. Leaf yellowing, oneof the most prominent features of plant aging may lead to reduced crop yields. The molecular mechanism ofresponses to senescence in soybean leaves is not completely clear. In our research, two soybean varieties wereselected with different stay-green traits: stay-green variety (BN106) and non-stay-green variety (KF14). RNAsamples extracted from the leaves of two varieties were sequenced and compared using high-throughputsequencing. Six key enzyme genes in chlorophyll degradation pathways were studied to analyze the changes intheir expression at seedling, flowering and maturation stage. Meanwhile, the construction of the genetictransformation process had been constructed to identify the function of putative gene by RNA-interference. Atotal of 4329 DEGs were involved in 52 functional groups and 254 KEGG pathways. Twelve genes encodingsenescence-associated and inducible chloroplast stay-green protein showed significant differential expression.MDCase and PAO have a significant expression in BN106 that may be the key factors affecting the maintenance of green characteristics. In addition, the function of GmSGRs has been identified by genetic transformation. The loss of GmSGRs may cause soybean seeds to change from yellow to green. In summary, ourresults revealed fundamental information about the molecular mechanism of aging in soybeans with differentstay-green characteristics. The work of genetic transformation lays a foundation for putative gene functionstudies that could contribute to postpone aging in soybeans

Advances in Agrobacterium tumefaciens-mediated transgenic cucumber / 生物工程学报

Cucumber (Cucumis sativus) is an important vegetable crop in the world. Agrobacterium-mediated transgenic technology is an important way to study plant gene functions and improve varieties. In order to further accelerate the transgenic research and breeding process of cucumber, we described the progress and problems of Agrobacterium tumefaciens-mediated transgenic cucumber, from the influencing factors of cucumber regeneration ability, genetic transformation conditions and various additives in the process. We prospected for improving the genetic transformation efficiency and safety selection markers of cucumber, and hoped to provide reference for the research of cucumber resistance breeding and quality improvement.

Improving crops genome through genetic engineering of the key metabolic pathways

Soybean loss due to pests and pathogens is a serious problem worldwide. Soybean producers have few options to manage diseases caused by general pathogens where major genes for full resistance have not been discovered. The innate defense of soybean plants could be enhanced by improving content and composition of lignin by genetic engineering of the phenylpropanoid pathway.We used a novel technique of germ-line genetic transformation of soybean plants via natural pollen tubes as vectors. This technique uses Agrobacterium tumefaciensto mediate transfer of genes of interest to the zygote to introduce the key lignification genes (PtMYB4, PAL5, F5H, CAD1) into soybean genome. We observed 5.6% average transformation efficiency in the first generation of transgenic plants and in the second generation the presence of the transgene constructs was confirmed in more than 50% (for CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1constructs) transgenic soybean lines. We confirmed the expression of the introduced genes at transcriptional level using RT-PCR and Northern blot. Functional analysis using lignin content determination and the activity of PAL5 and CAD1 enzymes demonstrated that the transgenes perform their function in planta. The proposed technique is effectiveand inexpensive and can be used to create novel stress and disease resistant soybean genotypes.

Cloning of AhWRKY33 from Asarum heterotropoides and its genetic transformation in Arabidopsis / 中国中药杂志

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.

A comparison of callus induction in 4 Garcinia species

Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation.

Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.

El frijol común en un cultivo recalcitrante a la regeneración in vitro y se carece de un protocolo eficiente y reproducible de transformación genética usando A. tumefaciens. Desarrollamos un protocolo de transformación genética mediada por A. tumefaciens de frijol común variedad Brunca utilizando genes marcadores (gusA y nptII) junto con el gen de la trehalosa-6-fosfato sintasa de levadura (TPS1) utilizado para incrementar tolerancia a estrés abiótico. La actividad de la β-glucoronidasa fue detectada en 45 % de los explantes infectados con la cepa LBA4404 de A. tumefaciens transformada con pCAMBIA1301. Después de 4 o 5 meses se regeneraron tallos en un medio adicionado con kanamicina. Los explantes supervivientes se evaluaron mediante PCR y presentaron una eficiencia de transformación de 0.5 %. El protocolo de transformación genética de frijol común establecido tiene dos ventajas adicionales con respecto a los reportes previos: (1) permite la obtención de regenerares transformados y (2) la transformación genética fue estable para el gen selectivo.

Rol genes enhance content of artemisinin and other secondary metabolites in Shennong hybrid of Artemisia annua / 中草药·英文版

Objective: Artemisia annua is the chief source of artemisinin, a potent antimalarial agent, in which other bioactive phytochemicals are also present. Due to low levels of bioactive compounds including artemisinin and flavonoids, it is necessary to increase the level of the secondary metabolites by regulating the expression of rol genes in the plant. Methods: A hybrid variety of A. annua (Hyb1209r, Shennong)developed by the Centre for Novel Agricultural Products, University of York, UK, was selected to produce transgenics of rolB and rolC genes. Genetic transformation was carried out via Agrobacterium tumefaciens GV3101 harboring rolB and rolC genes of Agrobacterium rhizogenes cloned separately. HPLC was used for the qualitative and quantitative analysis of flavonoids and artemisinin. Furthermore, thin layer chromatography (TLC)was also used to analyze artemisinin content. Results: Comparative analysis via HPLC revealed considerable enhancement in the phytochemical content of transgenic A. annua plants as compared to the wild type plant. Transgenics of rolB gene showed an average increase of 321% in rutin, 97.2% in caffeic acid, and 218.4% in myricetin, respectively. In the case of rolC gene transgenics, an average increase of 197.5% in rutin, 76.3% in caffeic acid, and 209.3% in myricetin was observed. Transgenics of rolB and rolC genes showed a 14.3%–28.6% and 2.8%–12.7% increase in artemisinin content respectively by HPLC analysis. TLC analysis showed that an average 142.2% and 110.2% enhancement in artemisinin for rolB and rolC transgenics respectively, compared with the wild type. An enhanced production of total flavonoids (average 30.2% and 25.5% increase in rolB and rolC transgenics, respectively)and total phenolics (average 34.3% and 25.8% increase in rolB and rolC transgenics, respectively)was observed as a result of transformation. Transformed A. annua plants showed improved free radical scavenging activity (average 46.5% and 29.1% increase in rolB and rolC transgenics, respectively)and total reducing power (average 32.7% and 26.4% increase in rolB and rolC transgenics, respectively)compared with untransformed plant. Conclusion: rolB and rolC genes were effective for developing A. annua plants with an enhanced level of phytochemicals.

Caracterización de indicadores de la calidad del fruto en líneas de fresa transgénicas con genes silenciados que codifican para enzimas pectinolíticas / Characterization of quality traits in transgenic strawberry fruits with genes encoding pectinolytic enzymes down-regulated

RESUMEN Se han evaluado algunos indicadores de calidad del fruto en líneas transgénicas de fresa con los genes de poligalacturonasa FaPGI (líneas PG) o pectato liasa FaplC (líneas APEL) silenciados. Se analizaron dos líneas independientes por genotipo transgénico. No se observaron diferencias en el contenido de sólidos solubles entre las líneas transgénicas y el control. De igual forma, la acidez total y el pH fueron similares en las líneas PG29, APEL21 y el control; sin embargo, la acidez de los frutos de las líneas PG62 y APEL39 fue superior al control. Los parámetros de color L*, a* y b* fueron similares en todos los genotipos; sin embargo, el contenido en antocianos fue menor en la línea APEL21. Los valores más altos de firmeza de fruto, estimada mediante un ensayo de extrusión, se observaron en las dos líneas transgénicas PG y en la línea APEL39. En cuanto a las pérdidas por goteo (drip loss), la línea APEL39 presentó un valor mayor que el control, pero la línea APEL21 registró valores menores. El contenido de compuestos fenólicos se analizó en la línea PG29, no encontrándose diferencias estadísticas con respecto al control. Finalmente, la capacidad del fruto para captar radicales libres fue ligeramente menor en la línea PG29 que en el control. Los resultados indican que el silenciamiento de los genes de pectinasas incrementa significativamente la firmeza de la fresa sin modificar sustancialmente parámetros de calidad del fruto maduro como color, acidez, sólidos solubles o contenido en antocianos.

ABSTRACT Some quality traits of transgenic strawberry fruits with low levels of expression of the pectinase genes FaPGI (PG lines) or FaplC gene (APEL lines) were evaluated. Two independent lines per transgenic genotype were analyzed. Soluble solids were similar in control and transgenic lines. Similarly, pH and titratable acidity was similar in lines PG29, APEL21 and control; however, lines PG62 and APEL39 showed acidity values higher than the control. The color parameters L*, a* and b* were similar in control and transgenic fruits; however, line APEL21 displayed a lower value of anthocyanin content. The highest values of fruit firmness, measured with an extrusion test, were observed in both PG transgenic lines and in the APEL39 line. Regarding the drip loss, APEL39 line showed a higher value than the control, but the APEL21 line displayed lower values. The content of phenolic compounds was analyzed in line PG29, not observing significant differences with the control. Finally, the antiradical activity of the fruit was slightly lower in the line PG29 than in the control. The results obtained indicate that the silencing of the pectinase genes increases the firmness of the fruit without substantially modifying other quality parameters such as color, acidity, soluble solids or anthocyanin content.

Establishment of a genetic transformation system for hairy root culture of Erigeron breviscapus / 中国中药杂志

The electroporation method was performed to transfer plasmid DNA of PBI-1300 carrying GFP gene into Agrobacterium rhizogenes C₅₈C₁ strains. Mediated by A. rhizogenes C₅₈C₁, the GFP gene were transformed into Erigeron breviscapus aseptic leaves by leaf disc method, then the hairy roots were induced and the infected hairy roots were screened by hygromycin resistance. The chromosomal DNA of the hairy root was used as the templates for the PCR amplification with the GFP-specific primers and then the expected amplified DNA bands appeared, the green fluorescent of GFP in the cut hairy roots was observed by two-photon microscope. These results indicated that GFP gene was integrated into the genome of E. breviscapus and was expressed stably. This study laid the groundwork for foreign gene high-efficiency expression inthe genetic transformation system for hairy root culture of E. breviscapus.

Establishment of transformation system of Claviceps purpurea Cp-1 strain / 药学学报

Claviceps pururea Cp-1 strain established in our lab is capable of producing variety of bioactive ergot alkaloids, and is broadly used by the pharmaceutical companies. To engineer the strain genetically for the production of specific ergot alkaloid, an effective transformation system must be set up first. However, the reported transformation system is not suitable for this strain due to different genetic backgrounds and the heterogeneity of Claviceps. Thus, in this paper, the hyphae of Cp-1 strain were used to prepare protoplasts by lywallzyme. The formation of protoplasts was investigated under different concentrations and incubation time of enzyme. The strain was tested for sensitivity to several antibiotics at different concentrations. Finally, the genetic transformation system of Cp-1 strain was established. The results suggest that protoplasts were formed efficiently by using 1% lywallzyme at 25℃ for 2 h. Transformants were obtained by PEG mediated protoplast transformation of Cp-1 strain with plasmid pAN7-1, using 1.5 mg·mL-1 hygromycin B as the selective marker. The exogenous gene in the plasmid pAN7-1 was integrated into the genome of Cp-1 strain transformant as demonstrated by PCR result. This study laid an important foundation for genetic manipulation of Cp-1 strain.

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.

Regeneração de plantas a partir de dois tipos de explantes de milho submetidos à transformação genética por biobalística / Regeneration of plants from two types of maize explants submitted to transformation by biolistic

O uso da biobalística na transformação genética de plantas requer a otimização de diversos parâmetros, entre eles, o desenvolvimento de protocolos para regeneração de plantas a partir de células transformadas. Este trabalho teve como objetivo avaliar a eficiência de regeneração de plantas de milho a partir de calos embriogênicos e de explantes sementes divididas, submetidos à transformação por biobalística, utilizando o gene bar como marcador de seleção. Foram submetidos 2000 agrupamentos de calos embriogênicos de milho e 2000 explantes sementes divididas à transformação por biobalística. A seleção dos eventos de transformação foi realizada em meios de cultivo suplementados com glifosinato de amônio. As plântulas selecionadas foram aclimatadas e transplantadas para vasos em casa de vegetação. Com a utilização de embriogênese somática, a eficiência de regeneração foi de 0,2%, enquanto que, com a utilização de sementes divididas, a eficiência de regeneração foi de 1,3%. Embora ambos os protocolos possibilitem a regeneração de plantas de milho submetidas à transformação por biobalística, há a necessidade de aumentar sua eficiência.

The use of biolistic on genetic transformation of plants requires an optimization of several parameters, including the protocol development for plant regeneration from transformed cells. The objective of this research was to evaluate the regeneration efficiency of maize plants from embryogenic callus and explants from split seeds submitted to transformation by biolistic using the gene bar as a selectable marker. It was submitted 2000 clusters of embryogenic callus and 2000 divided maize seeds to transformation by biolistic. The selection of transformation events was in culture medium supplemented with ammonium glifosinate. Selected seedlings were acclimatized and transplanted to pots in a greenhouse. Using somatic embryogenesis, the regeneration efficiency was 0.2%, while using split seeds explants the regeneration efficiency was 1.3%. Although both protocols allow the maize plant regeneration after transformation by biolistic, there is a need to increase its efficiency.