Sequence analysis and SSR marker mining of a peroxidase gene in Senecio scandens / 中草药

Objective: To study the relationship between the sequence structure and function of a peroxidase gene (POD) and mine and confirm SSR locus of this gene in Senecio scandens. Methods: A POD gene was obtained from full-length cDNA library of S. scandens. Then a series of online bioinformatics tools were used to analyze nucleic acid sequence and amino acid structure of this gene. According to its nucleotide sequence, one SSR locus was searched and a pair of primers were designed. The PAGE electrophoresis confirmed that this primer pair had polymorphism among several landraces. Results: The target gene had the structural characteristics of typical PODs. It showed a high conservation in functional domains. PAGE electrophoresis showed that the primer could be stably amplified in different landraces with obvious polymorphisms. Conclusion: The results expanded the understanding of the function and sequence of a POD in S. scandens. The newly developed marker could be effectively applied to the detection of POD gene and breeding projects, and it also provided reference for POD researches of other species.

Electrophoresis separation and MS analysis of proteins in fermentation broth of Poria cocos / 中草药

Objective: In order to carry out the study on proteins from Poria cocos fermentation broth, the proteins in the fermentation broth were separated and identified. Methods: Proteins were obtained by organic acid precipitation from the fermentation broth and the protein concentration was determined by Bradford method. The obtained P. cocos secreted proteins were separated on SDS-PAGE electrophoresis, subjected to in-gel digestion, then identified by mass spectrometric analysis followed by database searching. Results: The protein concentration in the fermentation broth was around 74.01 μg/mL, with the apparent molecular weight ranged from 2.8 × 104 to 1.3 × 105. A total of 52 P. cocos secreted proteins were identified, including catalase, protein kinase, alkaline protease, glucoamylase, lysozyme, and so on. Conclusion: P. cocos fermentation broth has abundant proteins, which could be a good material for the study of P. cocos protein and also a potential healthy food and beverage.

High-level expression of Actin of Paeonia lactiflora in Escherichia coli / 中草药

Objective: To construct the prokaryotic expression vector of Actin gene and to establish the high-level expression of Actin of Paeonia lactiflora in Escherichia coli. Methods: Based on cDNA sequence of Actin of P. lactiflora and the polyclonal sites on the prokaryotic expression vector pET-32a (+), a pair of PCR primers whose 5' end was inserted with BamH I and Hind III, respectively were designed. Subcloning of Actin was carried out using RT-PCR technique. The recombinant plasmid pMD18-T-PlActin and the prokaryotic expression vector pET-32a (+) was digested by BamH I and Hind III and the objective gene and the empty vector were purified. After ligation and transformation, the recombinant pET-32a (+)-PlActin, which was characterized by colony PCR, plasmid PCR, and double enzyme digestion, was transformed into BL21 (DE3), and the engineering expression strain was obtained. The expression of recombinant Actin protein in E. coli was induced at different concentration of inducer IPTG, different bacteria density, and different expression time. After SDS-PAGE and Coomassie brilliant blue R250 staining, the dry gel was prepared and the expression level of recombinant Actin protein was analyzed. Results: Subcloning sequencing result showed that the sequence of PlActin was exactly same with the objective sequence and the 5' and 3' ends were successfully inserted with BamH I and Hind III sites. The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin was constructed successfully. The best concentration of IPTG was 0.1 mmol/L and the bacteria density A600 was 0.4 to 0.6. The optimal expression time was 4 h. Conclusion: The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin is constructed and the high-level expression of Actin of P. lactiflora in E. coli is established.

Karyological and electrophoretic differences between Pomacea flagellata and P. patula catemacensis: Caenogastropoda: Ampullariidae

The widespread Mexican apple snail Pomacea flagellata (Say 1827) and the strictly endemic "tegogolo" P. patula catemacensis (Baker 1922) (restricted to Lake Catemaco), are the only known American Ampullariidae that have haploid complements n=13. Pomacea patula catemacensis has suffered a critical reduction in abundance due to immoderate fishing for human consumption. Chromosome slides were obtained from colchicine-injected Pomacea snails collected from nine locations along the coastal zone of the Gulf of Mexico, including Lake Catemaco, for use in principal component analysis (PCA). Total proteins in foot homogenates were analyzed through isoelectric focusing (IEF) and native-PAGE electrophoresis on polyacrylamide gels. The chromosome number 2n=26 was confirmed for snails from all locations, with a uniform 9 m + 4 sm formula. However, P. patula catemacensis showed significantly larger chromosomes (absolute size) than any population of P. flagellata. Pomacea patula catemacensis also differed from all populations of P. flagellata in a PCA with standardized data, i.e., independently of the absolute size difference between species. Proteins with an acid isoelectric point were dominant in the foot of both species. The electrophoresis analysis showed that P. flagellata has 17 protein bands, with an upper bound at IEF=7.6, while P. patula catemacensis has only 15 bands, with an upper bound at IEF=7 and a more evenly spaced band pattern. Molecular weights ranged from 40 to approximately 130 kDa in both species. Proteins with high values (>94 kDa) were the most abundant. Pomacea patula catemacensis showed a band of 93 kDa, which was absent from all specimens of P. flagellata. Samples of P. flagellata did not cluster according to any geographical pattern in the statistical analyses, nor did they show any taxonomically useful differences in their electrophoretic patterns that merit sub-specific discrimination.