Objective To discuss the SOE-PCR technique in long piece or multiple pieces gene mutations operation.Methods After the primary PCR reaction,besides putting the flanking primers of recommended concentration,we changed the traditional way into putting 100-time diluted internal primers which used to amplify the primary PCR templates.Through such operations,we can achieve the most satisfied templates concentration of SOE-PCR dynamically.Results Compare with the traditional SOE-PCR,two pieces combination products with the new way is more stable and specific while the specific abundance ratio keeps well.At the same time we achieve the three pieces combination with a total length of 4.2 kb in the same reaction while the specific amplification abundance also satisfied.Conclusion With the new method,we can overlap pieces combination of SOE-PCR in the same reaction tube with the specificity and abundance keeps well while not like the traditional way of combine two pieces together.When in multiple chimerical genes operation,this kind of method can reduce PCR reaction cycles,thus may be meaningful for chimerical genes operations.
The genus Porana s. l. was cladistically analyzed based on 33 morphological characters. Using the genus Cordisepalum as out group, 10 eaqually most parsimonious cladograms of Porana were yielded from cladistic analysis. The strict consensus tree showed that Porana s. l. was a natural taxon, and it was consistent with the results on pollen morphology and micromorphology of seed surface. Most taxonomists disagree to divide it into 4 or 5 genera proposed by some taxonomists. In the consensus tree consisted of two paratactic clades, one showed that subg. Porana and subg. Poranopsis have the sistergroup relationship, and in the other clade the subg. Tridynamia was resolved as sister to the subg. Dinetus. Further studies are needed before an unambiguous phylogeny is achieved for the broad sense genus.
Human P-glycoprotein encoded by the ATP-binding cassette sub-family B member 1 (ABCB1) gene is expressed in the blood-brain barrier. ABCB1 protects the brain from many drugs and toxins such as glucocorticoids through the efflux pump. Recent evidence suggests that a specific allele of the ABCB1 gene confers susceptibility to major depressive disorder (MDD) in the Japanese population. The aim of this study was to explore the association of ABCB1 gene polymorphisms with MDD in a local Chinese Han population.Two hundred and ninety-two MDD patients and 208 unrelated individuals were matched by age and sex and examined using a case-control design. Six single nucleotide polymorphisms (SNPs) of the ABCB1 gene, including rs1045642, rs2032583, rs2032582, rs2235040, rs1128503, and rs2235015, were genotyped by ligase detection reaction and multiplex polymerase chain reaction. Linkage disequilibrium and haplotype analysis were investigated in the two study groups.Significant protection for MDD individuals carrying the TG haplotype of rs1045642-rs2032582 was observed (odds ratio 0.470, 95% confidence interval 0.251-0.897, P=0.01). The rs2032582 (G2677T) and rs1128503 (C1236T) SNPs of ABCB1 showed nominal associations with MDD; the other four SNPs of the ABCB1 gene were not associated with MDD.Chinese individuals carrying the TG haplotype of rs1045642-rs2032582 had a nearly 53% lower risk of developing MDD. To the best of our knowledge, this is the first report to analyze the effect of ABCB1 polymorphism on the risk of MDD in a Chinese population.
In the ethylene signalling pathway, EIN2 is an essential signal transducer linking ethylene perception on endoplasmic reticulum to transcriptional regulation in the nucleus, EIN3/EIL1 transcription factors initiate various responses leading to an ethylene response, and EBFs function in ethylene perception by regulating EIN3/EIL1 turnover. In this work, we isolated and characterised three EIN3 homologs (CpEIN3a, CpEIN3b, and CpEIL1), and two EBF homologs (CpEBF1 and CpEBF2) from papaya fruits. During the fruit development, the transcripts of all these genes were accumulated at a low level before reaching a maximum in the third month, then showed a decrease. Exogenous ethylene application and 1-MCP treatment had different effects on the expression of these genes. The correlations between various physiological characteristics with the gene expressions showed that CpEIN3b, CpEIL1, and CpEBF1/2 were negative regulators in terms of papaya fruit ripening under exogenous ethylene treatment while CpEIN3a was a positive one, CpEIN3a was negatively correlated with the respiration rate and ethylene production of papaya fruits under 1-MCP treatment. It is suggested that CpEIN2, CpEIN3a/b, CpEIL1, and CpEBF1/2 are development- and ripening-related genes in papaya fruits. These results provide new insights into the understanding of the ethylene signalling cascade in papaya fruit development and ripening.
Objective To construct a new recombinant immunotoxin expression vector by fusing rat IFN-γ-inducible protein gene (rIP10) and a truncated pseudomonas exotoxin A ramification (PE38KDEL) gene, and explore the expression of the rIP-10-PE38KDEL fusion protein in NIH 3T3 cells. Methods rIP-10 was cloned by polymerase chain reaction (PCR). PE38KDEL gene was gained from an prokaryotic expression vector plasmid PRKL459K-IL-4-PE38KDEL by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pcDNA3.1(+). After the eukaryotic recombinant vector pcDNA3.1(+)-rIP-10-PE38KDEL was identified by PCR, restriction endonuclease digestion, and sequence analysis, then the vector was transfected into NIH 3T3 cells by liposome protocol. Immunofluorescence method was used to confirm the expression of the fusion gene in the NIH 3T3 cells. Results PCR, restriction endonuclease digestion, and sequence analysis revealed the rIP-10-PE38PE38KDEL fusion gene was cloned into the eukaryotic expression plasmid vector pcDNA3.1(+) successfully. The pcDNA3.1(+)-rIP-10-PE38PE38KDEL fusion gene could express in the NIH 3T3 cell. Conclusion The result provide the basis for research of the targeted cytotoxic activity to Th1 cell and may have some potential value in clinical application.
Chalcone synthase (CHS) catalyzes the first committed step for the biosynthesis of flavonoids, which are important for tobacco quality. To extend the knowledge on tobacco CHSs, we conducted comparative analyses of CHS genes among allotetraploid tobacco and its diploid progenitors. In this study, 8, 4, and 4 CHS genes were identified from N. tabacum, N. sylvestris and N. tomentosiformis, respectively. All the tobacco CHS genes contained one intron, and shared two highly conserved domains, Chal_Sti_Synt_N and ACP_Syn_Ⅲ_C. The putative polypeptides of tobacco CHSs contained 389 to 420 amino acids with predicted molecular weights ranging from 40.74 to 46.02 kDa. Sequence and phylogenetic analyses revealed that each NtCHS gene in the allotetraploid tobacco had one ortholog in both diploid genomes. The chromosome location analysis of NtCHSs suggested that CHSG and CHSJ genes were deduced to be closely linked with each other in tobacco genomes. The spatial expression analysis in three tobacco species showed the expression of all tobacco CHS genes exhibited lower levels in stems than those in leaves and roots, and some NtCHS homologous pair members also showed distinct expression patterns. The reproductive tissues had the highest accumulation of mRNA transcripts of NtCHS genes. In addition, most NtCHS genes were actively involved in the process of tobacco response to ABA, GA, MeJA, and many abiotic stresses including drought, salinity, darkness and phosphate starvation. This study provides new information about tobacco CHSs by revealing their real functions in tobacco flavonoids biosynthesis, as well as their resistance to abiotic stresses.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)