Identification and expression analysis of apple PDHB-1 gene family / 生物工程学报

Pyruvate dehydrogenase E1 component subunit beta-1 (PDHB-1) is a gene encoding the β-subunit of pyruvate dehydrogenase complex, which plays an important role in fruit acid accumulation. The aim of this study was to investigate the evolution characteristics of apple PDHB-1 family and its expression in apples with different acid contents. Bioinformatics analysis was performed using databases including NCBI, Pfam and software including ClustalX, MEGA, and TBtools. By combining titratable acid content determination and quantitative real-time PCR (qRT-PCR), the expression of this family genes in the peel and pulp of apple 'Asda' and 'Chengji No.1' with different acid content were obtained, respectively. The family members were mainly located in chloroplast, cytoplasm and mitochondria. α-helix and random coil were the main factors for the formation of secondary structure in this family. Tissue-specific expression profiles showed that the expression of most members were higher in fruit than in other tissues. qRT-PCR results showed that the expression profile of most members was consistent with the profile of titratable acid contents. In the peel, the expression levels of 14 members in 'Asda' apples with high acid content were significantly higher than that in 'Chengji No.1' apples with low acid content, where the expression difference of MdPDHB1-15 was the most significant. In the pulp, the expression levels of 17 members in 'Asda' apples were significantly higher than that in 'Chengji No.1' apples, where MdPDHB1-01 was the most highly expressed. It was predicted that PDHB-1 gene family in apple plays an important role in the regulation of fruit acidity.

Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia.

Background & objectives: Mycoplasma pneumoniae is the most important and common cause of community-acquired pneumonia (CAP). The conventional detection methods (culture and serology) lack sensitivity. PCR offers a better approach for rapid detection but is prone to carry over contamination during manipulation of amplification products. Quantitative real-time PCR (qRT-PCR) method offers an attractive alternative detection method. In the present study, qRT-PCR, PCR and serology methods were used to detect M. pneumoniae infection in cases of pneumonias and findings compared. Methods: A total of 134 samples consisting of blood (for serology) and respiratory secretions (for PCR and qRT-PCR) from 134 patients were collected. The blood samples were tested for IgG, IgM and IgA using commercially available kits. For standardization of PCR of M. pneumoniae P1 gene was cloned in pGEMTEasy vector. Specific primers and reporter sequence were designed and procured for this fragment. The qRT-PCR assay was performed to prepare the standard curve for M. pneumoniae positive control DNA template and detection in patient samples. Results: Of the 134 patients, 26 (19%) were positive for antibodies against M. pneumoniae. IgG was positive in 14.92 per cent (20) cases, IgM in 4.47 per cent (6) and IgA was positive in 5.22 per cent (7) cases. In the qRT-PCR assay 19 per cent (26) samples were positive. Of the 26 qRT-PCR positive samples, nine could be detected by serology. PCR was positive for 25 samples. An extra sample negative by PCR was detected by qRT-PCR. Thus, real-time PCR assay, PCR and serology in combination could detect M. pneumoniae infection in 43 patients. Interpretation & conclusions: The study shows that 17 patients were detected by serology alone, 17 were detected by qRT-PCR only and nine patients were positive by both serology and real-time PCR. Of the 134 samples tested, 25 were positive by conventional PCR, but qRT-PCR could detect one more sample that was negative by PCR and serology. These results suggest that a combination of two or three methods may be required for reliable identification of CAP due to M. pneumoniae.