Quantitation of PCR Products by Capillary Electrophoresis in a Single Run

A simple method was developed to quantify DNA fragments such as PCR (polymerase chain reaction) products by capillary electrophoresis. Restraint fragments with different lengths were employed as internal standards in the study, which makes it possible for the evaluation of the quantity of PCR product in a single run.

Cloning, bioinformatics and transcriptional analysis of caffeoyl-coenzyme A 3-O-methyltransferase in switchgrass under abiotic stress

Genes encoding enzymes involved in the lignin biosynthesis through phenylpropanoid pathway were not only associated with the lignin content, but also related to the abiotic stress resistances. As far as the production of liquid biofuels and cultivation within the marginal land are concerned, switchgrass could be the better candidate to determine the relationship between lower lignin content and physiological function under stress. Caffeoyl-coenzyme A 3-O-methyltransferase（CCoAOMT） is a key enzyme for the methylation reaction of lignin biosynthesis. For this purpose, we cloned a CCoAOMT gene from switchgrass and identified its expression patterns under abiotic stresses. The full-length CCoAOMT gene, designated PvCCoAOMT（Gen Bank accession no. KF041775）, was 1 005-bp in length, has an opening reading frame of 777 nucleotides encoding a 258-amino acid protein. The deduced amino acid sequence of PvCCoAOMT shared a high degree of similarity（up to 98%） with CCoAOMTs from Panicum virgatum allele（BAO20881）, Sorghum bicolor（XP002436550） and Zea mays（NP001131288）. Using quantitative real-time PCR（qRT-PCR）, the significant upregulation of PvCCoAOMT was observed in stem tissues at a later stage（24 h） after drought treatment, with the transcript level increasing 33-fold compared that of the controls. Moderate and insignificant inductions of PvCCoAOMT were also observed in both stems and leaves during the later stages after cold（48 h in stems, 12 h in leaves） and mechanical wounding（48 h in stems, 12 h in leaves） treatments, respectively. Our results showed the different expression patterns of PvCCoAOMT in drought, cold and mechanical wounding stresses. PvCCoAOMT can be highly induced by drought and cold stresses, which indicates that it may play a role in plant abiotic stress resistance, particularly in the regulation of drought and cold resistance.

A linear DNA template-based framework for site-specific unnatural amino acid incorporation

Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.