Southern Blot Analysis Research Articles

Age-associated telomere shortening in Thoroughbred horses.

Telomeres are genetically conserved repetitive terminal DNA that protect against genomic instability and shorten with ageing. Here, we reveal the leukocyte telomere length of Equus caballus by measuring terminal restriction fragments (TRFs) using Southern Blot analysis in a cohort of 43 Thoroughbred horses (age: 24 h-25 years). Heterogeneous TRFs were observed in each animal and large inter-animal variation in mean TRF was observed (range: 10.5-18.7 kbp). Mean TRFs were inversely correlated with age (r = -0.47). The estimated yearly rate of telomere attrition was 134 bp. Horses should be considered as an alternative animal model to investigate environmental and lifestyle factors that regulate telomeres and promote healthy ageing.

Improved protocol for the transformation of adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues

The production of transgenic citrus plants from adult tissues is difficult because of low regeneration and transformation rates. To increase the transformation efficiency of adult citrus tissues, an improved protocol involving adult Citrus sinensis Osbeck ‘Tarocco’ blood orange tissues was developed. Explants were pre-incubated in a liquid medium prior to infection by Agrobacterium tumefaciens. Plant materials were also incubated on callus-induction medium supplemented with various combinations of cytokinin (Cyt) and kanamycin (Kan). An appropriate pre-incubation of the explants increased the transformation efficiency of adult tissues. During the callus-induction period, the Cyt type and Kan concentration had the largest and smallest effects on the transformation efficiency, respectively. The most effective combination of plant growth regulator and Kan for the transformation of ‘Tarocco’ blood orange tissues was 2 mg L−1 2-isopentenyl adenine and 50 mg L−1 Kan. The transformation efficiency under the optimized conditions was 11.7%. A Southern blot analysis confirmed the integration of the transgene. These results indicated that the transformation efficiency of adult citrus tissues can be enhanced by optimizing the transformation conditions.

Genomic Sequence Analysis of the Multidrug-Resistance Region of Avian Salmonella enterica serovar Indiana Strain MHYL.

A series of human and animal diseases that are caused by Salmonella infections pose a serious threat to human health and huge economic losses to the livestock industry. We found antibiotic resistance (AR) genes in the genome of 133 strains of S. Indiana from a poultry production site in Shandong Province, China. Salmonella enterica subsp. enterica serovar Indiana strain MHYL had multidrug-resistance (MDR) genes on its genome. Southern blot analysis was used to locate genes on the genomic DNA. High-throughput sequencing technology was used to determine the gene sequence of the MHYL genome. Areas containing MDR genes were mapped based on the results of gene annotation. The AR genes blaTEM, strA, tetA, and aac(6′)-Ib-cr were found on the MHYL genome. The resistance genes were located in two separate MDR regions, RR1 and RR2, containing type I integrons, and Tn7 transposons and multiple IS26 complex transposons with transposable functions. Portions of the MDR regions were determined to be highly homologous to the structure of plasmid pAKU_1 in S. enterica serovar Paratyphi A (accession number: AM412236), SGI11 in S. enterica serovar Typhimurium (accession number: KM023773), and plasmid pS414 in S. Indiana (accession No.: KC237285).

Transformation of wheat Triticum aestivum with the HvBADH1 transgene from hulless barley improves salinity-stress tolerance

Studies have shown that the stress tolerance of cereal plants to osmotic or salinity stresses can be improved to varying degrees by the overexpression of an introduced betaine aldehyde dehydrogenase (BADH) gene. In the present study, the HvBADH1 gene from Hordeum vulgare L. var. nudum Hook. f., encoding a cytosolic BADH, was transferred into Triticum aestivum via traditional Agrobacterium tumefaciens-mediated transformation. Molecular methods, such as PCR, Southern blot analysis, and real-time quantitative RT-PCR were used to identify the successful integration and expression of the HvBADH1 transgene in genetically transformed wheat lines. To detect the efficacy of the HvBADH1 transgene in the transformants, some pivotal physiological indicators that reflected abiotic stress tolerance were measured in individual transgenic plant lines. These indicators included intracellular K+ and Na+ contents or K+/Na+ ratio, relative conductivity, and malondialdehyde and glycine betaine (GB) concentrations in cells. The results revealed that all the tested transgenic lines could significantly increase the recruitments of K+ in their cytosol than the wild-type seedlings. Similarly, 11.59- to 21.82-fold greater accumulation of GB, 2.11–2.56 times higher calli relative growth rates, and 26.2–29.1% seedling survival rates were found in transgenic lines under 150 mM NaCl stressed conditions. Our results demonstrated that by overexpressing the HvBADH1 transgene in genetically transformed wheat, the overall salt tolerance of the target plants was significantly increased, and the damaging effects of high salinity were significantly reduced.

Isolation and characterization of genes related to sheath blight resistance via the tagging of mutants in rice

Sheath blight disease (ShB) in rice is caused by Rhizoctonia solani. However, there have been no major genes identified from wild rice relatives and cultivated rice that encode resistance to R. solani. In this study, 300 Ds tagging mutants were generated via tissue culture regeneration methods from a Ds (dissociation) insertion line, and R. solani AG1-IA was inoculated to isolate the resistant mutants. The starter line that was homozygous (Ds-s) with a single Ds insertion in chromosome 1 exhibited a similar response to ShB as the non-transgenic plants. A Southern blot analysis showed that the Ds element was actively transposed in 300 R1 lines generated from Ds-s, and R2 plants were generated by selfing using 300 R1 lines with Ds insertions. Out of 300 R2 lines, two lines displayed susceptibility, and two lines exhibited strong resistance to ShB. In addition, 23 mutant lines displayed resistant symptoms, and the remaining 273 lines showed no differences compared to the ShB-susceptible wild-type control plants. Further examination revealed that the Ds gene was inserted at the second intron of either the PHYTOCHROME B (PHYB) or LAZY1 gene in two strong resistant mutants. We showed that the ethylene signaling genes (EIL1, EIL2, EIN2, and ERF63), jasmonic acid synthesis gene (LOX2), and salicylic acid signaling gene PBZ1 were up-regulated in the phyB mutants compared with the wild-type control that is susceptible to ShB. Further genetic study using EIL1 RNAi and EIL1 overexpression plants revealed that EIL1 positively regulates the resistance of rice to ShB. Overall, our analyses provided a basis for the identification of ShB resistance genes and the realization that PHYB negatively regulates the resistance of rice to ShB via the suppression of ethylene signaling.

Comparison of Southern-by-Sequencing (SbSTM) technology and Southern Blot Analysis for Molecular Characterization of Genetically Modified Crops

Southern blot analysis is typically used for molecular characterization of genetically modified (GM) crops. Southern-by-Sequencing (SbSTM technology, hereafter referred to as SbS) is a high-throughput, sequence-based alternative technique utilizing targeted sequence capture coupled with next-generation sequencing (NGS) and bioinformatics tools to achieve the same molecular endpoints. To demonstrate that both SbS and Southern blot analysis reach the same conclusions about insertion copy number and intactness of the inserted DNA, both techniques were used to characterize four soybean GM events containing simple or complex DNA insertions. To demonstrate that both techniques reach the same conclusions about the presence of unintended DNA, maize GM events containing Agrobacterium plasmid backbone fragments were characterized. Additionally, oligonucleotides containing varying lengths of target sequence were analyzed to compare both techniques’ sensitivity for detecting small insertions. SbS and Southern blots had similar sensitivity and provided comparable results for copy number and intactness of simple and complex DNA insertions. Both techniques also had comparable results for detection of unintended plasmid backbone DNA sequences and small DNA fragments. Thus, SbS can deliver the same endpoints as Southern blot analysis for key molecular characterization aspects of GM crops and gene edited varieties, providing important information to inform regulatory decisions.
 https://doi.org/10.21423/jrs-v07brink

A genetic system for biasing the sex ratio in mice.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ∼53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ∼4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.

Establishment of an efficient alfalfa regeneration system via organogenesis and the co-expression of Arabidopsis SOS genes improves salt tolerance in transgenic alfalfa (Medicago sativa L.)

The Salt Overly Sensitive (SOS) signal transduction pathway is pivotal in Na+ efflux and facilitates ion transport and homeostasis for improved salt tolerance in plants. Ten alfalfa varieties were used as experimental materials and two alfalfa regeneration systems were established and optimized. Cotyledons and hypocotyls were initially used as explants to induce embryogenic callus via the indirect production of somatic embryos to establish a callus acceptor system. Cotyledonary nodes were used as explants to induce adventitious bud formation via direct organogenesis, thereby establishing an in vitro regeneration system that could be used for the genetic transformation. Agrobacterium-mediated transformation of the cotyledonary nodes of the alfalfa ‘Golden Empress b’ was used to generate 25 independent sources of transformed plants exhibiting herbicide tolerance. Four of the positive transgenic plants were randomly selected for southern blot analysis, and three hybridization signals with one or two copies were detected. Reverse transcription polymerase chain reaction showed that the Bialaphos resistance (Bar) and SOS1 genes were expressed in transgenic plants and that multiple exogenous salt-tolerant genes were integrated into the transgenic plant genome and expressed at the transcriptional level. The overexpression of Arabidopsis SOS genes in alfalfa conferred a high degree of salinity tolerance, enhanced plant growth, lowered the accumulation of Na+, increased the accumulation of K+ in the leaves, and altered physiological and biochemical parameters in response to salt stress.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway

Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18µg/g FW) and its glycosylated form polydatin (33.6-67.15µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 106 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.

FvC5SD overexpression enhances drought tolerance in soybean by reactive oxygen species scavenging and modulating stress-responsive gene expression.

Overexpression of FvC5SD improves drought tolerance in soybean. Drought stress is one of the most important abiotic stress factors that influence soybean crop quality and yield. Therefore, the creation of drought-tolerant soybean germplasm resources through genetic engineering technology is effective in alleviating drought stress. FvC5SD is a type of C-5 sterol desaturase gene that is obtained from the edible fungus Flammulina velutipes. This gene has good tolerance to the effects of stresses, including drought and low temperature, in yeast cells and tomato. In this study, we introduced the FvC5SD gene into the soybean variety Shennong9 through the Agrobacterium-mediated transformation of soybean to identify drought-tolerant transgenic soybean varieties. PCR, RT-PCR, and Southern blot analysis results showed that T-DNA was inserted into the soybean genome and stably inherited by the progeny. The ectopic expression of FvC5SD under the control of a CaMV 35S promoter in transgenic soybean plants enhanced the plant's tolerance to dehydration and drought. Under drought conditions, the transgenic plants accumulated lower levels of reactive oxygen species and exhibited higher activities and expression levels of enzymes and cell than wild-type soybean. iTRAQ analysis of the comparative proteomics showed that some exogenous genes coding either functional or regulatory proteins were induced in the transgenic lines under drought stress. FvC5SD overexpression can serve as a direct and efficient target in improving drought tolerance in soybean and may be an important biotechnological strategy for trait improvement in soybean and other crops.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

TPP1 OB-fold domain protein suppresses cell proliferation and induces cell apoptosis by inhibiting telomerase recruitment to telomeres in human lung cancer cells.

Maintaining telomeres by recruiting telomerase-to-chromosome ends is essential for cancer cell survival. Inhibiting telomerase recruitment to telomeres represents a novel strategy for telomere-based lung cancer therapy. However, approaches for interrupting telomerase recruitment for cancer therapy still need to be explored. The telomere-binding protein TPP1 is responsible for recruiting telomerase to telomeres and synthesizing telomeres through the association between the oligosaccharide/oligonucleotide-binding (OB)-fold domain of TPP1 and telomerase reverse transcriptase. We overexpressed the TPP1 OB domain (TPP1-OB) by lentivirus infection in lung cancer cells. Telomere length was examined by Southern blot analysis of terminal restriction fragments. The effects of TPP1-OB on cell proliferation, the cell cycle, apoptosis, chemosensitivity, and tumor growth were evaluated in vitro and in vivo. TPP1-OB inhibited the recruitment of telomerase to telomeres and shortened telomere length by acting as a dominant-negative mutant of TPP1. TPP1-OB resulted in reduced cell proliferation, G1 cell cycle arrest, and increased cell apoptosis in lung cancer cells. Cell apoptosis occurred mainly through the caspase-3-dependent signaling pathway. TPP1-OB also suppressed anchorage-independent growth and tumor growth in vivo. Moreover, we demonstrated that TPP1-OB enhances the sensitivity of lung cancer cells to the chemotherapeutic drug paclitaxel. Our results suggest that inhibiting TPP1-mediated telomerase recruitment by expressing the TPP1-OB domain is a potential novel strategy for telomere-targeted lung cancer therapy.

Molecular characterization and infectivity analysis of a bipartite begomovirus associated with cotton leaf curl Multan betasatellite naturally infecting Rumex nepalensis in northern India

Rumex nepalensis (Nepal Dock) is an Indian traditional medicinal herb of the Western Himalayas. During a survey in 2013, typical begomovirus-like symptoms were observed on naturally grown R. nepalensis in the Bandla region of Palampur, Himachal Pradesh, India. PCR-based detection identified a bipartite begomovirus and a betasatellite in all the symptomatic plant samples. Complete sequence characterization established their identity as tomato leaf curl Palampur virus (ToLCPalV) and cotton leaf curl Multan betasatellite (CLCuMuB), respectively. Infectious clones of the virus and betasatellite were agro-infiltrated on both natural (R. nepalensis) as well as experimental (Nicotiana benthamiana) hosts. At 25 days post infiltration (dpi), N. benthamiana developed typical virus symptoms in DNA-A + DNA-B and DNA-A + DNA-B + CLCuMuB-infiltrated plants, whereas the plants infiltrated with DNA-A alone did not show any diseased phenotype. However, in combination with CLCuMuB, mild symptoms were observed. Agro-infiltrated R. nepalensis plants did not show visible symptoms in any of the above combinations. PCR and Southern blot analysis confirmed the replication and systemic spread of ToLCPalV and CLCuMuB in both R. nepalensis and N. benthamiana infiltrated plants. The present report shows R. nepalensis as a new natural host of the bipartite ToLCPalV associated with CLCuMuB. It also demonstrates the efficient trans-replication compatibility of CLCuMuB by a bipartite ToLCPalV in both of its natural and experimental hosts.

Prevention of mitochondrial genomic instability in yeast by the mitochondrial recombinase Mhr1

Mitochondrial (mt) DNA encodes factors essential for cellular respiration, therefore its level and integrity are crucial. ABF2 encodes a mitochondrial DNA-binding protein and its null mutation (Δabf2) induces mtDNA instability in Saccharomyces cerevisiae. Mhr1 is a mitochondrial recombinase that mediates the predominant form of mtDNA replication and acts in mtDNA segregation and the repair of mtDNA double-stranded breaks (DSBs). However, the involvement of Mhr1 in prevention of mtDNA deletion mutagenesis is unknown. In this study we used Δabf2 mhr1-1 double-mutant cells, which lose mitochondrial function in media containing fermentable carbon sources, to investigate whether Mhr1 is a suppressor of mtDNA deletion mutagenesis. We used a suppresivity assay and Southern blot analysis to reveal that the Δabf2 mutation causes mtDNA deletions rather than an mtDNA-lacking (ρ0) phenotype, and observed that mtDNA deletions are exacerbated by an additional mhr1-1 mutation. Loss of respiratory function due to mtDNA fragmentation occurred in ∆mhr1 and ∆abf2 mhr1-1 cells. However, exogenous introduction of Mhr1 into Δabf2 mhr1-1 cells significantly rescued respiratory growth, suggesting that Mhr1-driven homologous mtDNA recombination prevents mtDNA instability.

Overexpression of ICE1, a Regulator of Cold-Induced Transcriptome, Confers Cold Tolerance to Transgenic Zoysia japonica

Numerous genes are known to regulate the cold response in plants. The ICE1 gene is a regulator of coldinduced transcriptome playing an important role in cold tolerance in Arabidopsis thaliana. Here, the Arabidopsis ICE1 gene was cloned and transformed into A. thaliana and Korean lawn grass (Zoysia japonica) respectively, under the control of a maize ubiquitin promoter. The transgenic Arabidopsis plants showed higher survival rate, less MDA content, and higher levels of proline, SOD, and POD after freezing treatment. In the case of Z. japonica, 43 transgenic plants were produced by Agrobacterium-mediated transformation. Southern blot analysis showed that 9 transgenic lines selected by phosphinothricin (PPT) were transformed with ICE1 independently. Cold treatment significantly induced the transcripts abundant in the up-regulated ZjCBF and ZjDREB1 in transgenic lines OE-18 and OE-19. Meanwhile, transgenic plants showed increased proline level but decreased MDA content and with higher activities of SOD and POD compared with wild type under cold stress. These results suggest that overexpression of the ICE1 gene could improve the antioxidative capacity in conferring cold tolerance to the transgenic zoysiagrass plants.

Cloning and analysis of Ophiocordyceps xuefengensis mating type (MAT) loci.

The entomopathogenic fungus Ophiocordyceps xuefengensis, a recently described species and identified as the sister taxon of Ophiocordyceps sinensis, is a desirable alternative to O. sinensis. The mating systems of fungi play a vitally important role in the regulation of sexual reproduction and evolution, but the mating type loci of O. xuefengensis were completely unknown. In this study, the mating systems of O. xuefengensis were analyzed. The conserved α-box region of the MAT1-1-1 and HMG-box of MAT1-2-1 were successfully obtained by PCR amplification. The distribution of both mating types in different tissues of wild and cultivated O. xuefengensis growth was detected and analyzed. The results showed that the asci always harbored both mating types, whereas the sclerotium, the stipe and each isolated strain of wild O. xuefengensis always had only one idiomorph, either MAT1-1 or MAT1-2, which confirmed that O. xuefengensis is heterothallic. The MAT1-1 locus of O. xuefengensis harbors MAT1-1-1, MAT1-1-2 and MAT1-1-3, and MAT1-2 contains the MAT1-2-1 gene. Southern blot analysis showed the MAT-1-1-1 and MAT-1-2-1 genes were single-copy in O. xuefengensis. These results will help to understand its life cycle and support artificial cultivation of O. xuefengensis.

Production of Marker-Free Apple Plants Expressing the Supersweet Protein Gene Driven by Plant Promoter.

The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein.