AbstractUsing semi-quantitative reverse transcriptase polymerase chain reaction (semi-qRT-PCR), cell transfection, Hoechst 33342, acridine orange (AO) and flow cytometry methods, apoptotic changes in porcine pre-adipocytes treated with 9-cis retinoic acid (9-cisRA)–retinoid X receptor α (RXRα) ligand, transfected with pEnhanced green fluorescent proteins C2–RXRα (pEGFPC2–RXRα) or RXRα–small interfering RNA (RXRα–siRNA), respectively, were investigated. Observations of morphologic changes showed changes in cell structure, cellular shrinkage and condensation of chromatin, pyknosis of the nucleus into a compact body, complete fragmentation within the nuclear membrane, continuous budding and shedding, and, finally conversion of the cell into several apoptotic bodies wrapped in membranes of different sizes. Compared with the control group, the apoptotic rate was significantly lowered in the 10 nmol/l 9-cisRA-treated group, and significantly higher in the 10 μmol/l 9-cisRA-treated group (both P<0.05). Gene expression of RXRα was significantly up-regulated and down-regulated when cultured pre-adipocytes were transfected with pEGFPC2–RXRα or RXRα–siRNA (P<0.05), respectively; the apoptotic rate decreased in the pEGFPC2–RXRα group and significantly increased in the RXRα–siRNA group when compared with a control group (P<0.05). This implies that RXRα inhibits the apoptosis of porcine pre-adipocytes.
AbstractEpidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse (Mus musculus) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A431 carcinoma cells overexpressing EGFR on the surface, with an LD50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.
AbstractThe DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA ofPseudoalteromonas atlantica19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed inEscherichia coliER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.
AbstractSimple sequence repeat (SSR) analysis was performed on fiveSecalespecies, fourTriticumspecies and a Triticale line Fenzhi-1 using 102 pairs of microsatellite primers. A 387-bp specific DNA fragment FZ387 (GenBank accession no. EF179137) was obtained from the Triticale Fenzhi-1 with primer Xgwm614, without amplification inSecale. NCBI BLAST revealed that this FZ387 sequence had 94% and 95% similarity to part of the Gypsy Ty3-LTR retrotransposonFatimainTriticum monoccocum(AY485644) andTriticum turgidum(AY494981), respectively. A pair of specific polymerase chain reaction (PCR) primers, FaF and FaR, was designed based on the conserved region of this FZ387 sequence. The amplification of primer pair Xgwm614F and FaR revealed that a specific 350-bp band (designation as A350) was obtained from the species containing A chromosomes. Furthermore, PCR on Langdon Chinese Spring substitution lines was performed, and the results found that this segment was located on both long and short arms of all A chromosomes. However, the amplification of primer pair FaF and Xgwm614R gave rise to a specific DNA band of about 350 bp (designated AB350) from materials containing A and/or B chromosomes. The wild species of wheat and the relatives were amplified using the two pairs of primers, and revealed that only A350 and AB350 were found in Chinese Spring (CS). Sequence comparison and variation of SSR primers binding regions of FZ387 indicated that significant diversity might exist in the internal sequence of thisFatima-like element among triticeae genomes. Meanwhile, both A350 and AB350 can be used as molecular markers for the detection of A and AB genomes.
AbstractThis study aimed to explore high-throughput cDNA array monitoring technology and to apply it to the gene expression spectrum analysis of salinity-challenged tobacco plants. A Nicotiana tabacum cDNA library was sequenced and found to consist of 5927 high-quality sequences (GenBank accession nos CV015900-CV021826). By analysing the expressed sequence tags (ESTs), the proportion of N. tabacum genes was identified at the EST level. A cDNA array was constructed based on the tentative unique transcripts (TUTs) derived from EST assembling results. A total of 42 differentially expressed genes were identified, including plasma membrane intrinsic protein 2a, ethylene-responsive proteinase and pre-mRNA splicing factor prp31 gene, suggesting that there was a complicated biological response in N. tabacum under saline stress.
AbstractAn efficient AHL (N-acyl-homoserine lactone) bioassay strain, JZA1, of Agrobacterium tumefaciens was used to detect the AHL production from Acidovorax avenae subsp. citrulli [the pathogen causing bacterial fruit blotch (BFB) of melons], and the results showed that A. avenae subsp. citrulli produced a 3-O-C8-homoserine (HSL) type signal molecule. Gene aiiA, which could degrade AHL molecules, was transformed into A. avenae subsp. citrulli strain NJF10, creating strain NJF10-aiiA. The AHL production from NJF10-aiiA was significantly reduced compared with wild-type NJF10. Inoculation tests showed that NJF10-aiiA had an obvious reduction of virulence on watermelon fruits. Our finds showed that AHL production by A. avenae subsp. citrulli was related to its pathogenicity. This work might provide a novel way to control BFB by QS (quorum sensing) interference.
AbstractThe F1hybrid offspring of the distant hybridization betweenCichlasoma trimaculatum(male) andHeros managuense(female) were successfully obtained through artificial fertilization, and the chromosome specimen of kidney cells of the two parents and their offspring were prepared using a low colchicine concentration air drying method, Howell and Black's (1980) silver–nucleolar organizer region (Ag–NOR) staining method and Sumner's (1972) C-band staining method. The karyotypes, Ag–NORs and C-bands ofC. trimaculatum, H. managuenseand their offspring were observed. The results showed that the diploid numbers of the two parents and the F1hybrid offspring were all 2n=48. ForC. trimaculatum, the karyotype formula was 2n=2m+8sm+26st+12t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome st7, and the entire chromosomes of st9and st13and the centromeres of the other chromosomes had positive C-bands. ForH. managuense, the karyotype formula was 2n=2m+6sm+28st+12t, NF=56. Two Ag–NORs were located on the tips of the short arm of chromosome sm2and positive C-bands were found in the centromere of all chromosomes. In the F1hybrid offspring, the chromosome formula was 2n=2m+8sm+30st+8t, NF=58. Two Ag–NORs were seen on the tips of the short arm of chromosome sm2; all chromosomes presented positive C-bands in the centromere. No heteromorphic sex chromosome was found. After comparison with the two parents, the F1hybrid offspring was suggested to be the filial generation of the two hybrid parents rather than the gynogenesis ofH. managuenseinduced by heterogeneous sperm ofC. trimaculatum.
AbstractThe plant expression vectors pCAMBIA1301PMIand pBIPMIwere constructed by substituting theEscherichia coliphosphomannose-isomerase (PMI) gene for thehptgene of pCAMBIA1301 andgusgene of pBI121, respectively. Epicotyl explants of the Xuegan sweet orange (Citrus sinensisL. Osbeck) were inoculated withAgrobacterium tumefaciensEHA105- pCAMBIA1301PMIand EHA105-pBIPMIand subsequently selected in a medium supplemented with a combination of 25 g/l mannose and 5 g/l sucrose as the carbon source. The transformation efficiency rate was 27.7% when transformed by pCAMBIA1301PMIand 12.7% by pBIPMI. Genetic transformation was confirmed by chlorophenol red assay and polymerase chain reaction (PCR). A new method for obtaining transgenic Xuegan sweet orange plants was developed using thePMI/mannose selection system.
The potential relationship between the establishment of Foot-and-mouth disease virus (FMDV) persistent infection and gene variation was identified by investigating the variation of VP1 and 3ABC genes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×10 4 ID 50 /ml of FMDV O/Akesu/58 strain. After displaying clinical or subclinical signs, they probably became asymptomatic carriers. Oesophageal–pharyngeal fluids were collected monthly from the carriers with a probang and inoculated into a baby hamster kidney cell line (BHK-21); 12 FMDV isolates were obtained. The VP1 and 3ABC genes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of the VP1 nucleotide and amino-acid sequence of all the isolates was above 98%, with no base deletion or insertion. When compared with the O/Akesu/58 FMDV strain, the homology of the VP1 nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in the VP1 gene of the isolates, including 16 consistent nucleotide mutations, with only two of them leading to a change in amino acid (I 56 →T, A 210 →E). Moreover, it was found that four nucleotide points and three amino-acid points had transversions among all isolates. The 3ABC gene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in the VP1 and 3ABC genes, and was probably related to other structural protein gene and key factors.
AbstractDomestic yak (Bos grunniens) metallothionein-I (MT-I) and metallothionein-III (MT-III) cDNA 3′-end full-length sequences (331 and 378 bp; GenBank accession MT-I no. AY758557, MT-III no. DQ492300) from the total RNA of liver and brain tissues were amplified and cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid amplification of cDNA ends (RACE) using the primers YMT-I SP and M13 primers M4, and YMT-III SP and M13 primers M4, respectively, which included the MT-I (183 bp) coding sequence, MT-III (207 bp) cDNA coding sequences, and also the tailing signal AATAAA and Poly(A) at the 3′-ends of the MT-I and MT-III, respectively. The analyses showed that the yak cDNA sequence coding for MT-I protein was composed of 61 amino acids, including 20 cysteines having a conserved tripeptide structure, for example C–X–C, C–C–X–C–C, C–X–X–C and so on. The yak cDNA sequence coding for MT-III protein was composed of 68 amino acids, including 19 cysteines having both MT-III-specific conserved tripeptides and the same conserved tripeptides as MT-I, including T, CPCP, GEGAEA and so on. They were all comparatively conservative in molecular evolution. These structures indicated that MT-III had the same physiological functions regarding heavy metals (detoxification and so on) as MT-I, and also different specific functions from MT-I, including inhibition of neuron growth.
AbstractThe techniques used to detect genetically modified organisms (GMO), including qualitative polymerase chain reaction (PCR), quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and many others, are systematically described and discussed. The application progress of GMO in species-specific detection, endogenous genes, standard substances and restraining factors influencing detection are reviewed. The ongoing problems and development prospects of detection techniques of GMO are also pointed out.
AbstractThree pairs of primers were designed for amplification of the adiponectin gene in geese (Anser demeatica) based on the published sequences of chicken, duck and other adiponectin homologues. The complete 1062 bp sequence of the adiponectin gene in geese, obtained by polymerase chain reaction (PCR) amplification and splicing, contained two exons and one intron. The coding sequence was 738 bp long and included codes for 245 amino acids (GenBank Accession No. EU370686). The coding sequence and the corresponding protein of goose adiponectin shared the highest homology with ducks, 94.85% and 95.51%, respectively; homology with chicken adiponectin was next highest, while the lowest homology was to be found with mammals. The predicted molecular weight (MW) and isoelectric point (pI) were 26,603.3 Da and pH 5.19, respectively, which were highly similar to those of species considered above. Reverse transcription-PCR revealed that goose adiponectin mRNA was highly expressed in skeletal muscle, fatty tissue, heart and stomach muscles. Medium levels of expression were detected in the small intestine, stomach glandular tissue, kidney and lung, while it was only weakly expressed in the liver, spleen, ovaries and diencephalon.
AbstractThe expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.
Seven isoenergic semi-purified test diets containing graded levels of protein ranging from 20% to 50% were formulated using fish meal and casein as the protein sources. Triplicate groups of Barbudes caldwell juveniles with initial body weight of 1.26± 0.02 g respectively were fed by the test diets for 8 weeks. The results indicated that there was no significant effect of dietary protein levels on the survival rate, relative weight of viscera and relative weight of liver of the juvenile fish (P 0.05). The weight gain and specific growth rate of the fish were increased with dietary protein level increasing from 20% to 35% (P 0.05), but not affected significantly as dietary protein level increased from 35% to 50% (P 0.05). Feed efficiencies were not significantly different when fish fed diets with protein levels from 30% to 50%, but significantly higher than those of the other two treatments when fish fed diets with protein levels of 20% and 25% (P 0.05). The protein efficiency ratio(PER) was negatively correlated to the diet protein level(x) (PER = 3.006-0.03251x, R =0.9366). There was no significant effect of dietary protein levels on carcass moisture, crude protein and ash of the juveniles (P 0.05). While carcass lipid was decreased with the increase of dietary protein level (x) (L =8.2169-0.0458x, R =0.8551). There was no significant variation of hepato-pancreas protease activity among tests (P 0.05). Intestine protease activity and hepato-pancreas amylase activity were increased to some extent, and then decreased with continued increasing dietary protein level. The intestine amylase activity (IAA) of the juvenile was negatively correlated to dietary protein level (x) (IAA = 84.625-0.9147x, R =0.8463). It was estimated that the suitable protein level for the B. caldwell juvenile was 34% of dry diet when the broken-line model was introduced to regress the relationship of weight gain of the juvenile and dietary protein level.
AbstractThe genetic diversities of 72 individuals from three wild Lenok populations of Mudanjiang River (MD), Yalujiang River (YL) and Wusulijiang River (WSL) in the northeast of China were analysed using amplified fragment length polymorphism (AFLP) markers. The results showed that 541 polymorphic loci out of 559 were amplified by 12 primer pairs and the percentage of polymorphic loci was 96.78%. Shannon indices for the MD, YL and WSL populations were 0.3988±0.2913, 0.3254±0.3037, 0.2125±0.2862, respectively, and Nei's gene diversity indices were 0.2737±0.2062, 0.2229±0.2129, 0.1446±0.1985, respectively. The average total genetic diversity (Ht) was 0.3512±0.0.0208 and the average genetic diversity within populations (Hs) was 0.2137±0.0152. Among the three populations, the average genetic distance (Dst) was 0.1375 and the gene differentiation coefficient (Gst) was 0.3914. The genetic diversity was 60.85% within populations and 39.15% among populations. The gene flow index (Nm) was 0.7776. The analysis of molecular variance (AMOVA) indicated that the average fixation index (Fst) was 0.55336. The variance was 55.16% within populations and 44.84% among populations. The highest polymorphism ratio was in the MD group and the lowest in the WSL group.
AbstractA pair of degenerate primers was designed based on a conserved domain of GA 20-oxidase reported in other plants. The full-length (1179 bp) carnation (Dianthus caryophyllus L. cv. Master) GA 20-oxidase cDNA (named Dc20ox) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). BLAST analysis revealed that the deduced amino acid sequence had high homology (66–75%) with the GA 20-oxidase sequences from other plants. An RNAi vector (pART400) was constructed from a 400 bp fragment representing a highly conserved region of GA 20-oxidase.
The head kidney(HK)macrophages and the peripheral blood leukocytes were isolated from the head kidney and blood of Cyprinus carpio respectively by percoll gradient density centrifugation,cultured in vitro and exposed to different concentrations of lentinan.To evaluate the immunostimulating effects of lentinan,proliferation of the peripheral blood leukocytes,respiratory burst of macrophage and IL-1βmRNA expression of macrophages were investigated by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide),NBT(nitroblue tetrazolium)reduction,Griess reaction and real-time PCR,respectively.Results showed that proliferation of peripheral blood leukocytes after 24 h incubation by the induction of lentinan at 100 and 1000μg/mL was markedly stimulated.Lentinan at 1,100 and 1 000μg/mL could induce superoxide anion in macrophages markedly.Reduction of nitric oxide of HK macrophages after 96 h incubation lentinan showed it had no conspicuous effect on nitrogen burst activity of macrophages; moreover it inhibited the nitrogen burst activity at higher doses.The expression of IL-1βin the HK macrophages after 24 h stimulation by the polysaccharide showed lentinan stimulated IL-1βexpression in carp head kidney.It suggested that lentinan can modulate the immune response of C.carpio.
AbstractMature porcine oocytes containing first polar bodies (Pb I) were obtained byin vitroculture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown afterin vitrofertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h ofin vitromature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h afterin vitrofertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.
A rapid Real-time polymerase chain reaction (RT-PCR) for West nile virus(WNV) detection was established. Primers were designed according to Capsid protein gene by Primer Premier5.0. In response, a cheap assay with the intercalating dye SYBR green Ⅰ was developed and validated. Amplifying curve showed that this method could successfully amplify 102 copies/μL WNV gene, meanwhile reference Japanese encephalitis virus(JEV) and blank control were all negative. Ten-fold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay showed high reproducibility with CV2%, indicating that the developed RT-PCR assay, a rapid, sensitive and specific test, has been built for detecting WNV.
AbstractSeventeen special loci were selected from 77 microsatellite markers to distinguish three varieties of tilapias, including the six differential loci UNH636, UNH117, UNH172, UNH738, UNH878 and UNH896 in Oreochromis aureus; five differential loci UNH913, UNH907, UNH222, UNH980 and UNH880 in O. niloticus; and six differential loci of UNH876, UNH899, UNH853, UNH932, UNH933 and UNH773 in O. mossambicus. Any one of the 17 loci could amplify particular bands to distinguish one tilapia from the other two. The genetic structure of O. aureus, O. niloticus and O. mossambicus stocks and their phylogenetic relationships were also analysed using these 17 loci. In total 142 alleles were detected, and the average number of alleles per locus was 8.35. Additionally, a clustering analysis was performed based on the result of the Popgen32 software package and phylogenetic trees were constructed by MEGA4 using the unweighted pair group method using arithmetic average (UPGMA). The results showed that the mean value of observed heterozygosity was 0.0941, 0.5490 and 0.2588, the mean value of expected heterozygosity was 0.1089, 0.7230 and 0.1965, and the polymorphism information content was 0.0869, 0.7149 and 0.1643, in O. aureus, O. niloticus and O. mossambicus, respectively. The UPGMA tree demonstrated that O. aureus was more closely related to O. mossambicus than to O. niloticus.
