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L. Viridiana Soto-Robles María Fernanda Lpez Vernica Torres-Banda Claudia Cano-Ramírez Gabriel Obregn-Molina Gerardo Zúiga 《International journal of molecular sciences》2021,22(1)
Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree. 相似文献
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Nina Franko Lucija Ana Vr
aj Taja Zore Barbara Ostanek Janja Marc Jasna Lojk 《International journal of molecular sciences》2022,23(8)
RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases. 相似文献
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Longjian Niu Yan-Bin Tao Mao-Sheng Chen Qiantang Fu Chaoqiong Li Yuling Dong Xiulan Wang Huiying He Zeng-Fu Xu 《International journal of molecular sciences》2015,16(6):12513-12530
Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi. 相似文献
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Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content 总被引:1,自引:0,他引:1
Victor Ladero Daniel M. Linares María Fernndez Miguel A. Alvarez 《Food research international (Ottawa, Ont.)》2008,41(10):1015-1019
Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the Ct values and histamine concentrations determined by HPLC. 相似文献
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