Rapid detection of strawberry mottle virus using reverse transcription recombinase polymerase amplification with lateral flow strip.

Strawberry mottle virus (SMoV) is one of the main RNA viruses that profoundly affects the growth of strawberries worldwide. The rapid on-site detection of SMoV described here can be applied to produce virus-free strawberry seedlings. Reverse transcriptase recombinase polymerase amplification (RT-RPA) was combined with lateral flow (LF) strip to rapidly detect SMoV. The detection limit was 500 fg of RNA under optimized conditions. The SMoV-RT-RPA-LF assay was optimal with a combination of 2 µL reverse primer (5 µM) and 0.6 µL probe (10 µM) in a 50 µL RT-RPA reaction mixture for isothermal amplification at 40 â„ƒ for 15 min. In addition, 100 suspected samples were collected from different regions in the Shanghai suburbs. The SMoV-RT-RPA-LF assay showed that 3 of these 100 samples were positive for SMoV, which was in good concordance with the reverse transcription polymerase chain reaction (RT-PCR) results. The primers and probe had a unique specificity to SMoV because there was no cross-reaction with other strawberry viruses. This study provides an effective technique for the rapid on-site detection of SMoV to ensure a virus-free strawberry nursery.

Genome-wide identification of expansin in Fragaria vesca and expression profiling analysis of the FvEXPs in different fruit development.

Strawberry is a highly efficient and economical horticultural crop plant, and strawberry fruits are easy to soften after ripening and decay after harvest, which severely impacts the economic benefits. Expansins are plant cell-wall loosening proteins involved in the process of fruit softening, loosening cell walls and reducing fruit firmness. In this study, 35 FvEXPs genes were identified in the F. vesaca genome. These genes were divided into four subfamilies (27 FvEXPAs, 5 FvEXPBs, 1 FvEXLAs, and 2 FvEXLBs) and were unevenly distributed on 7 chromosomes. Gene structure and motif analysis showed the conserved structure and motif in same subgroup, however, the different motifs and structures may reveal functional divergence of multigene family members of FvEXPs in different developmental stages of fruits. The expression profiling by RNA-seq and qRT-PCR analysis revealed that the FvEXP genes have distinct expression patterns among different stages of strawberry development and ripening. Among them, 3 genes (FvEXPA9, FvEXPA12, and FvEXPA27) were highly expressed in the ripening stage, FvEXPA9 and FvEXPA12 were especially highly expressed in turning stage, whereas FvEXPA27 was especially highly expressed in red stage. Our study provides a better understanding of the FvEXP genes, which may benefit strawberry biotechnological breeding and genetic modification for improving fruit quality and delaying fruit softening.

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry.

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. 'Shanghai Angel' by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of 'Shanghai Angel' and other cultivars to improve agronomic traits.

Ram semen preserved at 0°C with soybean lecithin Tris-based extender substituted for egg yolk.

OBJECTIVE: The present study evaluated the preservation of ram semen at 0°C using soybean lecithin with a Tris-fructose extender. METHODS: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0°C in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination. RESULTS: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05). CONCLUSION: These results suggest that ram sperm is capable of fertilization after preservation at 0°C with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

The sugar transporter system of strawberry: genome-wide identification and expression correlation with fruit soluble sugar-related traits in a Fragaria × ananassa germplasm collection.

Sugar from plant photosynthesis is a basic requirement for life activities. Sugar transporters are the proteins that mediate sugar allocation among or within source/sink organs. The transporters of the major facilitator superfamily (MFS) targeting carbohydrates represent the largest family of sugar transporters in many plants. Strawberry (Fragaria × ananassa Duchesne) is an important crop appreciated worldwide for its unique fruit flavor. The involvement of MFS sugar transporters (STs) in cultivated strawberry fruit sugar accumulation is largely unknown. In this work, we characterized the genetic variation associated with fruit soluble sugars in a collection including 154 varieties. Then, a total of 67 ST genes were identified in the v4.0 genome integrated with the v4.0.a2 protein database of F. vesca, the dominant subgenome provider for modern cultivated strawberry. Phylogenetic analysis updated the nomenclature of strawberry ST homoeologs. Both the chromosomal distribution and structural characteristics of the ST family were improved. Semi-RT-PCR analysis in nine tissues from cv. Benihoppe screened 34 highly expressed ST genes in fruits. In three varieties with dramatically differing fruit sugar levels, qPCR integrated with correlation analysis between ST transcript abundance and sugar content identified 13 sugar-correlated genes. The correlations were re-evaluated across 19 varieties, including major commercial cultivars grown in China. Finally, a model of the contribution of the sugar transporter system to subcellular sugar allocation in strawberry fruits was proposed. Our work highlights the involvement of STs in controlling strawberry fruit soluble sugars and provides candidates for the future functional study of STs in strawberry development and responses and a new approach for strawberry genetic engineering and molecular breeding.

Draft Genome Sequence of the Strawberry Anthracnose Pathogen Colletotrichum fructicola.

Colletotrichum fructicola is a causal agent of strawberry anthracnose and a major economic pathogen of horticultural and ornamental crops worldwide. Here, we present an annotated draft genome sequence for a C. fructicola isolate previously used for transcriptomic analysis. The assembly totals 58.0 Mb in 477 contigs with 18,143 predicted genes.

First record of Leptospira and Blastocystis infections in captive flying squirrels (Trogopterus xanthipes) from Enshi County, China.

In traditional Chinese medicine, the feces of flying squirrels have long been used to promote blood circulation and relieve bodily stasis. However, the excrement of flying squirrels may harbor zoonotic agents that could be hazardous to public health. To understand the occurrence of bacterial and parasitic infections in this species, we investigated selected zoonotic pathogens including Leptospira and Blastocystis in the urine and feces of flying squirrels in China. Urine and fecal samples from flying squirrels were collected from a family-owned flying squirrel farm located in Enshi County, Hubei Province in China. Leptospira and Blastocystis DNA was extracted from the urine and feces of flying squirrels, and used as targets for PCR amplification, using different specific primers. PCR amplification and DNA sequencing showed that 4.4% (3/69) of flying squirrels were positive for Leptospira, while 30.4% (21/69) of the animals were positive for Blastocystis. Notably, 1.4% (1/69) of flying squirrels were found to be co-infected with Leptospira and Blastocystis. Sequence analyses allowed for the detection of 3 Blastocystis subtypes (ST1, ST3 and ST13), and mixed infections of Blastocystis subtype 1 and subtype 3 were found in 4.4% (3/69) of flying squirrels. Phylogenetic analysis of the 16S ribosomal RNA gene (rrs2), the flagellin B gene (flaB), and outer membrane lipoprotein lipL32 gene (LipL32) sequences indicated that the Leptospira species detected in the study was L. interrogans. We concluded that flying squirrels from central China were infected with Leptospira and Blastocystis, suggesting that these animals can be a source of infection for their owners, and using fresh excrement from this animal as traditional medicine could be risky to human health. To the best of our knowledge, this is the first report of Leptospira and Blastocystis infection in flying squirrels from Enshi County, China. Our findings provide new data on the epidemiology of these pathogens in this region.

Ehrlichia species in pond-farmed leeches (Hirudinaria sp.) in Hubei Province, China.

Leeches are frequently used in traditional Chinese medicine. However, they are potentially hazardous to human and animal health by transmitting several pathogens. Studies of diseases transmitted by leeches are scarce. The purpose of this study was to analyze the pathogens carried in pond-farmed medicinal leech in China. Leeches were collected from 6 farms in Hubei Province in central China. DNA was extracted from the internal organ of leeches to analyze the origin of blood meal. Leech genera were confirmed through amplification of 18S rRNA and mitochondrial gene cytochrome oxidase I (COI) gene by PCR and host animal species were identified through amplification of mitochondrial cytochrome b gene. Species of Ehrlichia in the leech specimens were screened with PCR using specific primers. PCR amplification and DNA sequencing showed that 620 leeches were Hirudinaria sp. Ehrlichia DNA was detected in 39 specimens from 2 farms. We obtained a total of 65 sequences of the cytB gene from 620 leech internal organ samples including sequences of human (n = 5), rat (n = 1), domestic pig (n = 10), duck (n = 23), goose (n = 12) and buffalo (n = 14). Phylogenetic analysis of the rrs and groEL gene sequences showed that Ehrlichia detected in the study were closely related to Ehrlichia sp. in ticks from Korea and Japan. To the best of our knowledge, this is the first report on Ehrlichia DNA being detected from leeches. Our findings provided new data on Ehrlichia spp. and farmed leech species in China.

Cloning, Characterization, and Expression Analysis of a Gene Encoding a Putative Lysophosphatidic Acid Acyltransferase from Seeds of Paeonia rockii.

Tree peony (Paeonia section Moutan DC.) is an excellent woody oil crop, and the cloning and functional analysis of genes related to fatty acid (FA) metabolism from this organism has not been reported. Lysophosphatidic acid acyltransferase (LPAAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. This project reports a putative lysophosphatidic acid acyltransferase gene PrLPAAT1 isolated from Paeonia rockii. Our data indicated that PrLPAAT1 has 1047 nucleotides and encodes a putative 38.8 kDa protein with 348 amino acid residues. Bioinformatic analysis demonstrated that PrLPAAT1 contains two transmembrane domains (TMDs). Subcellular localization analysis confirmed that PrLPAAT1 is a plasma membrane protein. Phylogenetic analysis revealed that PrLPAAT1 shared 74.3 and 65.5% amino acid sequence identities with the LPAAT1 sequences from columbine and grape, respectively. PrLPAAT1 belongs to AGPAT family, and may have acyltransferase activity. PrLPAAT1 was ubiquitously expressed in diverse tissues, and PrLPAAT1 expression was higher in the flower and developing seed. PrLPAAT1 is probably an important component in the FA accumulation process, especially during the early stages of seed development. PrLPAAT1 overexpression using a seed-specific promoter increased total FA content and the main FA accumulation in Arabidopsis transgenic plants.

The different interactions of Colletotrichum gloeosporioides with two strawberry varieties and the involvement of salicylic acid.

The disease symptoms recognized as 'Anthracnose' are caused by Colletotrichum spp. and lead to large-scale strawberry (Fragaria×ananassa Duchesne) losses worldwide in terms of both quality and production. Little is known regarding the mechanisms underlying the genetic variations in the strawberry-Colletotrichum spp. interaction. In this work, Colletotrichum gloeosporioides (C. gloeosporioides) infection was characterized in two varieties exhibiting different susceptibilities, and the involvement of salicylic acid (SA) was examined. Light microscopic observation showed that C. gloeosporioides conidia germinated earlier and faster on the leaf surface of the susceptible cultivar compared with the less-susceptible cultivar. Several PR genes were differentially expressed, with higher-amplitude changes observed in the less-susceptible cultivar. The less-susceptible cultivar contained a higher level of basal SA, and the SA levels increased rapidly upon infection, followed by a sharp decrease before the necrotrophic phase. External SA pretreatment reduced susceptibility and elevated the internal SA levels in both varieties, which were sharply reduced in the susceptible cultivar upon inoculation. The less-susceptible cultivar also displayed a more sensitive and marked increase in the transcripts of NB-LRR genes to C. gloeosporioides, and SA pretreatment differentially induced transcript accumulation in the two varieties during infection. Furthermore, SA directly inhibited the germination of C. gloeosporioides conidia; NB-LRR transcript accumulation in response to SA pretreatment was both dose- and cultivar-dependent. The results demonstrate that the less-susceptible cultivar showed reduced conidia germination. The contribution of SA might involve microbial isolate-specific sensitivity to SA, cultivar/tissue-specific SA homeostasis and signaling, and the sensitivity of R genes and the related defense network to SA and pathogens.

Nucleotides maintain the activity of Cav1.2 channels in guinea-pig ventricular myocytes.

The activity of Cav1.2 Ca(2+) channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca(2+) channels in the presence of 1 µM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca(2+) channels.

Low-Mg(2+) treatment increases sensitivity of voltage-gated Na(+) channels to Ca(2+)/calmodulin-mediated modulation in cultured hippocampal neurons.

Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry.

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry (Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

Family-wide expression characterization of Arabidopsis beta-carbonic anhydrase genes using qRT-PCR and Promoter::GUS fusions.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes found throughout the phylogenetic tree. The ß-class carbonic anhydrases (ß-CAs) are the predominating class of CAs in plants. Growing evidence underscores the importance of ß-CAs in plant immunity and environmental adaptation in addition to their roles in photosynthesis. However, many fundamental problems in Arabidopsis ßCAs expression remain unsolved. Here we examined the transcript abundance of AtßCAs in different tissues of Arabidopsis thaliana, and the accumulation of mRNA in response to CO2 and darkness. Histochemical analysis was performed to study the promoter activity of AtßCAs during post-germination seedling growth and in mature plants. All six members of the AtßCA subfamily showed a response to changed CO2 level and darkness, but each member showed a specific dynamic pattern. Although expression of each AtßCA was unique, in general most AtßCAs were synchronously expressed in green leaves since 5 days after germination until flowering. AtßCA1 and AtßCA2 were most highly expressed in leaves but AtßCA2 displayed weaker expression in roots. The level of AtßCA3 transcripts was highest in flowers, while AtßCA5 was most widely expressed and might be involved in more processes than other members. AtßCA6 was unique for increased expression in darkness and no expression in either the anther or pistil. The present study provides useful information for further functional investigation.

Genome-wide identification and comparative expression analysis of NBS-LRR-encoding genes upon Colletotrichum gloeosporioides infection in two ecotypes of Fragaria vesca.

Anthracnose caused by Colletotrichum spp. is one of the most destructive diseases of cultivated strawberry (Fragaria×ananassa Duchesne) worldwide. The correlation between NBS-LRR genes, the largest class of known resistance genes, and strawberry anthracnose resistance has been elusive. BLAST search in NCBI identified 94 FvNBSs in the diploid genome of strawberry Fragaria vesca, with 67 of the TIR-NBS-LRR type. At least 36 FvNBSs were expressed, with 25% being non-coding genes. Two F. vesca ecotypes, HLJ and YW, showed great variations in both morphological and physiological responses upon C. gloeosporioides infection. qRT-PCR revealed that 5 of the 12 leaf-expressed FvNBSs displaying opposite transcription responses to C. gloeosporioides infection in two ecotypes. These results showed that the transcriptional responses of several FvNBSs were involved in the ecotype-specific responses to C. gloeosporioides in F. vesca. These FvNBSs hold potential in characterizing molecular components and developing novel markers associated with anthracnose resistance in strawberry.

Isolation and characterization of two YUCCA flavin monooxygenase genes from cultivated strawberry (Fragaria × ananassa Duch.).

UNLABELLED: In strawberry (Fragaria × ananassa Duch.), auxin has been recognized as the main signal molecule coordinating the growth and initiation of ripening of fruits. The molecular mechanism regulating auxin biosynthesis in strawberry remains unknown. This project reports two YUCCA flavin monooxygenase genes FaYUC1-2 isolated from cultivated strawberry. FaYUC1 and FaYUC2 are most homologous to AtYUC6 and AtYUC4, respectively. Significant expression of FaYUC1-2 is found in vegetative meristems and reproductive organs, with overlapping but distinct patterns. During fruit development, both transcripts of FaYUC1 and FaYUC2 in achenes reach a peak around large green fruit (G2) stage, but the sudden rise in FaYUC2 transcript level is much steeper and begins earlier than that in FaYUC1. FaYUC2 is also obviously expressed in the receptacles from green fruits, hinting another auxin source for receptacle development, other than achenes. FaYUC1 over-expression Arabidopsis exhibits typical auxin hyper-accumulation phenotype in many aspects, such as the narrow and downward curled leaves, strong apical dominance, short and hairy root. It is also severely sterile, due to the disruption of floral meristems initiation and floral organs development. Transgenic analysis indicates that strawberry YUC gene may hold conserved role in auxin biosynthesis like their homologs in other plants. Integrated with the spatiotemporal expression features, these results led us to propose that FaYUC1-2 may involve in many developmental processes including flower and fruit development in strawberry. KEY MESSAGE: This paper is the first report of isolation and characterization of strawberry auxin biosynthesis genes. And their conserved functions in auxin biosynthesis were confirmed after ectopic expression.

Molecular diversity and differential expression of starch-synthesis genes in developing kernels of three maize inbreds.

The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various grain traits. To explore the unique mechanism controlling the advantageous waxy trait and characterize the molecular feature of genes relevant to starch composition in two elite waxy inbreds, expression profiling combined with gene organization analysis was performed in them as compared to one normal inbred. Genotype-specific expression patterns were observed for most genes studied. The waxy inbreds were shown to contain mutations in multiple starch-synthesis genes, namely gbssI (wx), gbssIIb and isa2 (potentially isa3 too).The mis-splicing events directly accounted for wx loss of function. Contrarily, disruption of 5' and 3' transcript sequence may contribute to the absence of GbssIIb and Isa2 transcripts in waxy inbreds, respectively. Besides, the splicing of Sugary1 transcript was developmentally regulated in the normal inbred, and DNA polymorphisms were detected within SSIIIb-1 gene in waxy inbreds.

Attenuation of streptomycin ototoxicity by tetramethylpyrazine and its effect on K⁺ channels in the outer hair cells of guinea pig cochlea.

In order to elucidate the mechanism underlying the attenuation of streptomycin ototoxicity by tetramethylpyrazine (TMP), the present study investigated the effect of TMP on the outward K(+) current in the outer hair cells of guinea pig cochlea. Sixty guinea pigs were divided into 6 groups randomly. Auditory brainstem response (ABR) was used to observe the change in thresholds and to evaluate ototoxicity induced by streptomycin. Whole-cell patch-clamp technique was used to observe the effect of TMP on outward K(+) current in isolated outer hair cells. The results showed that TMP attenuated the threshold shift caused by streptomycin and increased the amplitudes of Ca(2+)-sensitive K(+) current [I(K(Ca))] in the outer hair cells. The present data suggest that TMP displays anti-ototoxicity induced by streptomycin. The augmented amplitudes of I(K(Ca)) of the outer hair cells induced by TMP may be one of the mechanisms underlying its ototoxicity-attenuating effect.

[Study on parentage testing in JIRONG rabbit by microsatellite markers].

Thirteen microsatellite markers, including Sat2,Sat3,Sat4,Sat5,Sat7,Sat8,Sat12, Sat13, Sat16,Sol08,Sol28,Sol30 and Sol03, were studied for their parentage-testing application in a group of 30 JIRONG Rabbits in the present report. The 13 microsatellite loci were successfully amplified with specific primers designed according to known sequences. The PCR products amplified from the microsatellite loci were analyzed by 8% denaturing polyacrylamide gel electrophoresis. The results demonstrated that the average alleles and the mean heterozygosity (Hs) and the polymorphism information content (PIC) and the combined exclusion probability (PE2) of the 13 microsatellite loci were 3.46, 0.578, 0.531, and 0.999329, respectively. The exclusion probability (PE1) of the 13 loci was 0.935226, and the confidence of the parentage testing was less than 80% when data of both parents were unknown, while the exclusion probability (PE1) and the confidence were 0.999329 and 95% respectively with known data of a single parent. Since the data of the rabbit maternal lines studied were known, the paternal lines of the group were successfully identified using the 13 microsatellite loci with high confidence.

[Study of ceramide monohexoside in ovarian cell line COC1/DDP resistant to cisplantin].

OBJECTIVE: To investigate the effect of ceramide monohexoside (CMH) on resistance to cisplatin and apoptosis in ovarian cell line COC1/DDP, and to provide new ideals and clues to seek new effective methods for studying the mechanism and reversing the resistance in ovarian cell line as well. METHODS: COC1 cells and COC1/DDP cells (before and after the treatment of mifepristone) were collected and neutral glycosphingolipids (N-GSLs) of the cells was isolated and purified, changes of CMH content were analyzed by high performance thin layer chromatography (HPTLC). The COC1/DDP cells were divided into three groups, one treated by cisplatin, one treated by mifepristone, the other treated by cisplatin and mifepristone. The survival rate of cells in three groups were evaluated by the methyl thiazolyl tetrazolium (MTT) assay, DNA ladders were presented by DNA gel electrophoresis, the forms of cells were observed by transmission electron microscope (TEM). RESULTS: The levels of CMH were (37.1 +/- 3.3)% in COC1/DDP, higher than that in COC1 (14.1 +/- 1.4)% (P < 0.001). After treating by 1.25, 5 micro mol/L mifepristone, the CMH were (26.6 +/- 2.6)% (P < 0.05) and (17.5 +/- 0.7)% (P < 0.001), respectively. Mifepristone had no effect on the viability of COC1/DDP cell below a concentration of 5 micro mol/L. But when mifepristone of 1.25 or 5 micro mol/L combined with cisplatin at a concentration of 0.1, 0.25, 0.5, 1.25, 2.5 micro g/ml, the inhibition rate of COC1/DDP cell is higher than that of COC1/DDP cells only treated by cisplatin at the concentration of 0.1 to 2.5 micro g/ml (P < 0.001). The combined treatment elicited DNA fragmentation, however, neither cisplatin of 1.25 micro g/ml nor mifepristone of 5 micro mol/L alone could potentiate DNA fragmentation. After the combined treatment, the COC1/DDP cells produced apoptosis body. CONCLUSIONS: CMH is related with resistance to cisplantin in ovarian cell line COC1/DDP. When CMH of COC1/DDP cells was inhibited by mifepristone, the cells were sensitive to cisplatin and apoptosis was elicited.