Browsing by Subject "gene expression"

Obesity and insulin resistance (IR) are key factors lead to equine metabolic syndrome and laminitis. Diet may play an important role in eliciting obesity by affecting insulin dynamics. Insulin-pathway signaling and mTORC1 genes may contribute to incred IR. The first objective of this study was to find and validate internal control genes for quantitative PCR method for adipose tissues in Finnhorse mares. The second aim was to quantitate the expression of mTORC1 and insulin-pathway associated genes after pasture season in two different treatment groups of Finnhorse mares and compare gene expression differences between treatment groups. In addition, gene expression differences were compared between two different adipose tissues. Twenty-two mares were equally divided into eleven equal pairs, the two mares of each group were randomly grazed either on cultivated high-yielding pasture (CG) or on semi-natural grassland (NG) from the end of May to the beginning of September. Eight pairs of Finnhorse mares were selected for gene expression profiling. Subcutaneous adipose tissue (SAT) samples were collected from two groups of Finnhorse mares after pasture season. Gene expression of neck and tailhead SAT were determined with quantitative Real-Time PCR method (qPCR). The selected internal control genes were actin beta (ACTB), glucuronidase beta (GUSB) and mitochondrial ribosomal protein L39 (MRPL39). Candidate genes were mechanistic target of rapamycin (MTOR), sterol regulatory element binding transcription factor 1 (SREBF1), sterol regulatory element binding transcription factor 2 (SREBF2), TBC1 domain family member 7 (TBC1D7), leptin (LEP), glucose transporter type 4 (GLUT4), monocyte chemoattractant protein-1 (MCP-1), retinol binding protein 4 (RBP4), tuberous sclerosis 1 (TSC1), tuberous sclerosis 2 (TSC2). There were no distinct gene expression differences between NG and CG groups in both neck and tailhead SAT. However, RBP4 had significantly (P=0.035) higher and GLUT4 had a trend (P=0.064) to higher mRNA expression in CG group in neck SAT. TSC1 had a trend (P=0.071) of higher expression in CG group in tailhead SAT. Gene expression differences were observed between tailhead and neck SAT. SREBF1 and GLUT4 had significantly (P=0.007 and P=0.026, respectively) higher expression levels in tailhead SAT compared to neck SAT. RBP4 had a trend (P=0.066) to higher expression in neck SAT compared to tailhead SAT. Minor differences in gene expression between NG and CG groups indicate that pasture-associated fat depositionmaynotconsiderably affect expressionof insulin-pathway and mTORC1 genes associated to obesity and IR in studied subcutaneous adipose tissues. These results also provide additional evidence to our hypothesis that fattening resulting on unrestricted grazing on cultivated high-yielding pasture does not increase the risk of metabolic diseases in Finnhorse mares when they have normal body condition at the beginning of the grazing season.

Fragaria x ananassa is a widely appreciated berry with its production growing all around the world. Thus, there will be a huge demand for strawberry breeding in the future especially since the climate change is casting an extra shadow upon the growing conditions which is also why there is a need for better understanding of different cultivar types. There are everbearing and seasonally flowering cultivars of both Fragaria x ananassa and Fragaria vesca. The seasonally flowering types such as ʻHapilʼ flower once during the growing season whereas everbearing types such as ʻCalypsoʼ flower for a longer period. The gene behind the change in the flowering habit has been tracked to the photoperiodic pathway of F. vesca although the gene behind the trait in F x ananassa remains unknown. The aim of this project was to compare flowering and vegetative responses of in vitro propagated everbearing F x ananassa cultivar ʻCalypsoʼ and seasonally flowering ʻHapilʼ in long and short day photoperiodic conditions in order to find out differences between everbearing and seasonally flowering cultivars. This was done by collecting data from phenotype observations linked to the vegetative and generative stages of the development of strawberries. The phenotype data was then combined with gene expression data of FaSOC1, FaTFL1, FaGA20ox4 and FaAP1 which are genes known to work on the photoperiodic pathway that regulates the switch between the vegetative and generative development of both F x ananassa and F. vesca. In addition, the expression of an everbearing phenotype associated gene FaFT2 was analysed. This study was a part of a larger project aimed to find out the genetic basis for the everbearing habit of F x ananassa. Part of the ʻCalypsoʼ plants were induced to flower already during the acclimatization period and the rest at the very beginning of the treatment period which then caused differential flowering times between the ʻCalypsoʼ groups. Short day grown ʻHapilʼ was induced to flower between weeks three and six whereas long day grown ʻHapilsʼ remained vegetative. Phenotypic observations were also backed up by the expression of FaTFL1 and FaAP1. Instead the FaSOC1 expression was repressed in short day conditions more than in the long days regardless of the cultivar type. ‘Calypsos’ were capable of producing runners regardless of photoperiod or flower induction. Consequently the runner production seemed to be regulated by factors outside of the photoperiodic pathway. However, the expression of runnering associated FaGA20ox4 was low and variable due to the sampling strategy. Interestingly most of the axillary meristems of short day grown ‘Hapils’ remained dormant for an unknown reason. Expression of FaFT2 was low on the apical meristems and further support for the role of the gene in everbearing phenotype was not found.

Aluminum (Al3+) toxicity is a major limiting factor in acidic soils when pH<5.5 and faba bean experiences yield decreases in these conditions. The multidrug and toxic compound extrusion (MATE) family plays a vital role in Al3+ tolerance across species. This study searched for an ortholog of MtMATE66, a gene involved in Al3+ resistance in barrel medic, in faba bean and quantified the ortholog’s relative expression in 4 faba bean genotypes (GPID_0022, GPID_0153, GPID_0178 and GPID_0191). pBLAST of MtMATE66 in the faba bean unpublished genome identified the ortholog jg20333.t1, with 88.6 % identity, e-value 0.0 and bit score 880. InterPro Scan and NCBI CDD conserved domain queries classified jg20333.t1 as a MATE in the DinF subfamily. MEME Suite identified the 50-amino acid citrate exuding motif characteristic to MATEs exuding citrate in Al3+ tolerance while a multiple sequence alignment and phylogenetic analysis clustered jg20333.t1 with Al3+ -tolerant citrate exuding MATEs. The SWISS Model 3D structure and DeepTMHMM predicted an α-helical, twelve-transmembrane protein topology. The plasma membrane was predicted as the subcellular localisation of jg20333.t1 by ProtComp, WoLFSPORT and YLoc, however SignalP identified no signal peptides. The molecular weight 54.57kDa, theoretical isoelectric point 8.60 and grand average of hydropathicity 0.67 of jg20333.t1 were calculated by ProtParam. EMBOSS Needle and GSDS aligned 13 exons. Four biological replicates of faba bean plants were set up for 3 different treatments: acidic of pH 3.81 (Ac), acidic of pH 3.81 plus Al3+ (Al) and neutral of pH 6.01 (N) in a greenhouse and root tip samples were collected 45 days after transplanting in peat media for RNA extraction. The relative expression of jg20333.t1 was determined by RT-qPCR of jg20333.t1 as target gene, Vfactin as reference gene and N as internal calibrator. The Cq values generated were analysed using the 2-ΔΔCq method and showed high relative fold change in both Ac and Al. The upregulation in Al confirmed the implication of jg20333.t1 in faba bean tolerance to Al3+. The upregulation in Ac suggests upstream regulation by STOP transcription factor. The four genotypes had no significant difference in fold change. Based on these results, it is concluded that jg20333.t1 is a faba bean MATE gene, VfMATE, implicated in Al3+ tolerance by citrate exudation.

The main objective of this thesis was to analyse the impact of dry period energy intake on the intensity of metabolic stress and insulin resistance through changes in gene expression in dairy cows. Calving and beginning of lactation cause a negative energy balance which increases fat mobilization, metabolic stress and insulin resistance. These are intensified by high milk yield and excess energy intake from feed during the dry period. 14 genes were selected for the analysis. These genes encode proteins that are linked to lipogenesis, lipolysis and insulin signaling. The changes in the gene expression of subcutaneous adipose tissue were compared between two groups of eight cows. The control group was fed according to the energy requirements while the test group was fed ad libitum. The biopsies were taken -8d, +1d and +9d from parturition, cDNA was synthesized from them and they were analysed by quantitative PCR and 2-??CT-method. There Expression differences were observed in seven genes within the whole data between biopsy dates. During the period from before to after parturition, differences were identified in the expression of 11 genes within the whole data and the control group, and one gene in the test group. The most significant differences were observed in LEP and SCD genes. Between the groups there was a difference in the gene expression of IRS1 before parturition. This refers to the test group being more insulin resistant. After parturition there were no significant differences found in the gene expressions. The most significant changes observed in this study were as expected in early lactation. In the period between before and after parturition there were bigger changes in the gene expression observed in the control group than in the test group. The results indicate that cows getting more energy from their feed during the dry period are more insulin resistant before parturition.