Hairy root induction and plant regeneration of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> Libosch. f. <Emphasis Type="Italic">hueichingensis</Emphasis> Hsiao via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255.     

Identification and polymorphism of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> complex

Pectinase (endo-polygalacturonase) is the key enzyme splitting plant pectin. The corresponding single gene PGU1 is documented for the yeast S. cerevisiae. On the basis of phylogenetic analysis of the PGU nucleotide sequence available in the GenBank, a family of divergent PGU genes is found in the species complex S. bayanus: S. bayanus var. uvarum, S. eubayanus, and hybrid taxon S. pastorianus. The PGU genes have different chromosome localization.     

Resveratrol Synthase Transgene Expression and Accumulation of Resveratrol Glycoside in <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis>

Rehmannia glutinosa L. is an important medicinal crop in Asian countries and contains trace amount of resveratrol compounds. To increase production of the compounds, we attempted ectopic expression of peanut resveratrol synthase gene (AhRS3) in R. glutinosa. The AhRS3 sequence that encompassed the open reading frame, including a 312 bp-long intron present between the 59th and 60th codon, was driven by the CaMV35S promoter and introduced into R. glutinosa via Agrobacterium-mediated transformation of leaf explants. The transgenic plants with one to three copies of AhRS3 transgene showed normal growth and development. The transgene was expressed constitutively in the leaf, root and flower at similar levels. Transgene expression in the leaf resulted in the production of new compounds identified as resveratrol and 3′-H-resveratrol-3-O-β-d-glucoside (R-gluc) through nuclear magnetic resonance spectroscopy and mass spectrometry. R-gluc accumulated predominantly and its content in the leaf of the 11 transgenic lines ranged from 22 to 116μg/gFW. The contents of resveratrol compounds in the transgenic plants were further increased by cold, UV, ethylene, and paraquat treatments, and were positively associated with the levels of AhRS3 mRNA levels. The R-gluc isolated from the transgenic plants exhibited antioxidant activity equivalent to one-third of resveratrol. Transgenic plants were highly resistant to Fusarium oxysporum infection. The results indicate that the ectopic expression of AhRS3 in R. glutinosa results in the production of R-gluc and resveratrol at hundreds of times higher levels than in peanut seed. The increased production of resveratrol compounds from R. glutinosa, which show diverse benefits for human and plant health, could provide a new opportunity for the improvement of R. glutinosa products.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Modelling the pollen season start in <Emphasis Type="Italic">Corylus avellana</Emphasis> and <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Hazel (Corylus avellana L.) and black alder (Alnus glutinosa (L.) Gaertn.) are important sources of airborne pollen and represent an allergen threat during the flowering period. Researches on airborne pollen concentrations in both species are useful in allergology, as well as for fruit production for hazel. The aims of the present study were: (1) to investigate the relationships between environmental conditions and the airborne pollen concentration of hazel and black alder during the flowering period by correlation and multiple regression analysis and (2) to predict the pollen season start (PSS) by using a sequential model, in order to obtain a helpful tool in allergology and hazel cultivation. In this study, the applied method defines the pollen season as the period in which 90 % of the total season’s catch occurred, using a data set of 18 years (1996–2014). The relationships between daily meteorological parameters (temperature, humidity, rainfall and wind speed) during the 14-day period that precedes the PSS and the PSS of hazel and black alder (day of the year) were investigated. The results showed that mean temperature and the number of rainy days before the PSS are the main factors influencing PSS for both taxa. Moreover, the chilling and heat needed to break dormancy were estimated in order to predict the PSS of both species. Different years and different thresholds of temperature and chill days were used to calibrate and validate the model.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Identification,isolation and expression analysis of eight stress-related <Emphasis Type="Italic">R2R3</Emphasis>-<Emphasis Type="Italic">MYB</Emphasis> genes in tartary buckwheat (<Emphasis Type="Italic">Fagopyrum tataricum</Emphasis>)

Key message

Eight R2R3 - MYB genes in tartary buckwheat were identified, and their expression patterns were comprehensively analyzed, which reveals role in plant response to abiotic stresses.

Abstract

The proteins of the R2R3-MYB superfamily play key roles in the growth and development processes as well as defense responses in plants. However, their characteristics and functions have not been fully investigated in tartary buckwheat (Fagopyrum tataricum), a strongly abiotic resistant coarse cereal. In this article, eight tartary buckwheat R2R3-MYB genes were isolated with full-length cDNA and DNA sequences. Phylogenetic analysis of the members of the R2R3-MYB superfamily between Arabidopsis and tartary buckwheat revealed that the assumed functions of the eight tartary buckwheat R2R3-MYB proteins are divided into five Arabidopsis functional subgroups that are involved in abiotic stress. Expression analysis during abiotic stress and exogenous phytohormone treatments identified that the eight R2R3-MYB genes responded to one or more treatments. This study is the first comprehensive analysis of the R2R3-MYB gene family in tartary buckwheat under abiotic stress.

     

Cloning of two cellobiohydrolase genes from <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> and heterogenous expression in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml⁻¹ under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.