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1.
In this work we have demonstrated two independent real-time PCR methods for detection single nucleotide polymorphism (SNP) of genes csn and acyl-CoA: diacylglycerol acyltransferase 1 (dgat) in cattle. We have analyzed 296 samples of milk production cattle of Ukrainian breeding. The genotype frequencies were AA--0.58, AB--0.34, BB--0.08 for csn gene and for dgat gene--AA--0.7, AK--0.26, KK--0.04. High efficiency of so called "anti-primer" method was shown. Duration of anti-primer PCR reaction was about 2-2.5 hours only and provided full investigation of unknown gene allele.  相似文献   

2.
Increasing the oil concentration in soybean seeds has been given more attention in recent years because of demand for both edible oil and biodiesel production. Oil concentration in soybean is a complex quantitative trait regulated by many genes as well as environmental conditions. To identify genes governing seed oil concentration in soybean, 16 putative candidate genes of three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl-CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) involved in triacylglycerol (TAG) biosynthesis pathways were selected and their sequences retrieved from the soybean database (http://www.phytozome.net/soybean). Three sequence mutations were discovered in either coding or noncoding regions of three DGAT soybean isoforms when comparing the parents of a 203 recombinant inbreed line (RIL) population; OAC Wallace and OAC Glencoe. The RIL population was used to study the effects of these mutations on seed oil concentration and other important agronomic and seed composition traits, including seed yield and protein concentration across three field locations in Ontario, Canada, in 2009 and 2010. An insertion/deletion (indel) mutation in the GmDGAT2B gene in OAC Wallace was significantly associated with reduced seed oil concentration across three environments and reduced seed yield at Woodstock in 2010. A mutation in the 3′ untranslated (3′UTR) region of GmDGAT2C was associated with seed yield at Woodstock in 2009. A mutation in the intronic region of GmDGAR1B was associated with seed yield and protein concentration at Ottawa in 2010. The genes identified in this study had minor effects on either seed yield or oil concentration, which was in agreement with the quantitative nature of the traits. However, the novel gene-specific markers designed in the present study can be used in soybean breeding for marker-assisted selection aimed at increasing seed yield and oil concentration with no significant impact on seed protein concentration.  相似文献   

3.
The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.  相似文献   

4.

An oligonucleotide microarray—which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis—was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples.

The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.  相似文献   

5.

Background

The purpose of this study was to evaluate the effects of eight single nucleotide polymorphisms (SNP), previously associated with meat and milk quality traits in cattle, in a population of 443 commercial Aberdeen Angus-cross beef cattle. The eight SNP, which were located within five genes: μ-calpain (CAPN1), calpastatin (CAST), leptin (LEP), growth hormone receptor (GHR) and acylCoA:diacylglycerol acyltransferase 1 (DGAT1), are included in various commercial tests for tenderness, fatness, carcass composition and milk yield/quality.

Methods

A total of 27 traits were examined, 19 relating to carcass quality, such as carcass weight and fatness, one mechanical measure of tenderness, and the remaining seven were sensory traits, such as flavour and tenderness, assessed by a taste panel.

Results

An SNP in the CAPN1 gene, CAPN316, was significantly associated with tenderness measured by both the tenderometer and the taste panel as well as the weight of the hindquarter, where animals inheriting the CC genotype had more tender meat and heavier hindquarters. An SNP in the leptin gene, UASMS2, significantly affected overall liking, where animals with the TT genotype were assigned higher scores by the panellists. The SNP in the GHR gene was significantly associated with odour, where animals inheriting the AA genotype produced steaks with an intense odour when compared with the other genotypes. Finally, the SNP in the DGAT1 gene was associated with sirloin weight after maturation and fat depth surrounding the sirloin, with animals inheriting the AA genotype having heavier sirloins and more fat.

Conclusion

The results of this study confirm some previously documented associations. Furthermore, novel associations have been identified which, following validation in other populations, could be incorporated into breeding programmes to improve meat quality.  相似文献   

6.
Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.  相似文献   

7.
Fang X  Zhang J  Xu H  Zhang C  Du Y  Shi X  Chen D  Sun J  Jin Q  Lan X  Chen H 《Molecular biology reports》2012,39(2):1801-1807
Diacylglycerol acyltransferase (DGAT) plays a critical role in the synthesis of triacylglycerol. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variations of DGAT-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of DGAT-2 gene were investigated, only exon 3 of DGAT-2 gene showed polymorphism. The alignment between nucleotide sequences of NM_205793.2 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (A????G) in exon 3 of DGAT-2 gene, which resulted in amino acid change (Lys????Arg) and constructed two genotypes (AA, AB). The frequencies of allele A and genotype AA were dominant in all three breeds. And there was no significant difference for genotypic and allelic frequencies among the three breeds. The genotype distributions were in good agreement with Hardy?CWeinberg equilibrium (P?>?0.05) in each breed. Significant statistical differences were only found in withers heights (P?<?0.05) in Xuhuai goat between genotypes. The results indicated that individuals with genotype AA were significantly higher than those of individuals with genotype AB in withers height (P?<?0.05). No polymorphism was detected in the intron 3, exon 8 and 3?? flanking region. So we suggested that DGAT-2 gene had the close relationship with growth traits in goats. And this mutation could be used as a perfect molecular marker for marker-assisted selection (MAS) in animal genetics and breeding.  相似文献   

8.
We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT). Its expression in Saccharomyces cerevisiae resulted in an increase both in triacylglycerol content and in microsomal oleyl-CoA:diacylglycerol acyltransferase activity. Such effects were similar to those of characterized plant DGAT genes. This is the first DGAT gene isolated from an invertebrate. The phylogenetic relationships between DGATs and animal and yeast acyl-CoA:sterol acyltransferases are illustrated.  相似文献   

9.
An oligonucleotide microarray-which allows for parallel genotyping of many SNPs in genes involved in cow milk protein biosynthesis-was used to identify which of the 16 candidate SNPs are associated with milk performance traits in Holstein cows. Four hundred cows were genotyped by the developed and validated microarray. Significant associations were found between four single SNPs, namely DGAT1 (acyloCoA:diacylglycerol acyltransferase), LTF (lactoferrin), CSN3 (kappa-casein), and GHR (growth hormone receptor) and with fat and protein yield and percentage. Many significant associations between combined genotypes (two SNPs) and milk performance traits were found. The associations between the combined genotypes DGAT1/LTF and DGAT1/LEPTIN analyzed traits are presented as examples. The microarray based on APEX (Arrayed Primer Extension) is a fast and reliable method for multiple SNP analysis of potential application in marker-assisted selection. After further development, the chip may prospectively be used for dairy cattle paternity analysis and evolutionary studies.  相似文献   

10.
The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.  相似文献   

11.
The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.  相似文献   

12.
In yeast, the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE) occurs mainly in mitochondria. CL and PE have overlapping functions, and they are required for mitochondrial function. PE is physiologically linked with triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, involving an acyl-CoA-independent pathway through the phospholipid:diacylglycerol acyltransferase activity of the Lro1 protein. There is no report on the physiological link between CL and TAG metabolism. Here we report a metabolic link between CL and TAG accumulation in the S. cerevisiae. Our data indicated that CL deficiency causes TAG accumulation, involving an acyl-CoA-dependent pathway through the diacylglycerol acyltransferase activity of the Dga1 protein with no changes in the TAG molecular species. The DGA1 gene deletion from the CL-deficient strains reduced the TAG levels. Data from in vitro and in vivo analyses showed that CL did not affect the enzymatic activity of Dga1. Our data also showed that CL deficiency leads to the up-regulation of acetyl-CoA synthetase genes (ACS1 and ACS2) of the cytosolic pyruvate dehydrogenase bypass pathway. This study establishes a physiological link between CL and TAG metabolism in S. cerevisiae.  相似文献   

13.

Background

Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco.

Methods

Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested.

Results

Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001).

Conclusion

Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.  相似文献   

14.
The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.  相似文献   

15.
The activity of diacylglycerol acyltransferase of a subcellular particulate fraction from maturing safflower seeds was remarkably stimulated by the addition of 1, 2-diacylglycerols which were previously emulsified in a gelatin solution by sonication. Metal ions were inhibitory to the reaction. Deoxycholate and diisopropyl fluorophosphate were the most effective inhibitors. Sulfhydryl groups seemed to be of limited significance in the enzyme. Both 1, 2-dioleoyl-sn-glycerol and 2, 3-dioleoyl-sn-glycerol were good substrates of diacylglycerol acyltransferase, but the 1, 3-isomer did not serve as an acyl acceptor. The enzyme showed broad specificity for synthetic rac-1, 2-diacylglycerols containing various fatty acids. However, rac-1, 2-diacetylglycerol and rac-1, 2-dibutyrylglycerol, which are soluble in water, were ineffective. The enzyme exhibited no significant specificity for saturated and unsaturated fatty acyl-CoA thioesters as acyl donors. This suggests that the fatty acid composition at the 3-position of the glycerol molecule of safflower triacylglycerols may depend on the composition of the endogenous acyl-CoA pool.  相似文献   

16.
In this study, a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)-like protein is identified and isolated from the diatom microalga Phaeodactylum tricornutum (PtDGAT3). Analysis of the sequence reveals that ptDGAT3 cDNA encodes a protein of 504 amino acids with a molecular mass of 64.5 KDa. The putative ptDGAT3 protein has two catalytic domains: a wax ester synthase-like acyl-CoA acyltransferase domain and a bacteria-specific acyltransferase domain, which shows higher similarity to the DGAT3 of Acinetobacter calcoaceticus than reported DGAT1 or DGAT2 from high plants or algae. Its activity was confirmed by heterologous expression of PtDGAT3 in a neutral lipid-deficient quadruple mutant yeast Saccharomyces cerevisiae H1246. The recombinant yeast restored the formation of a lipid body and displayed a preference to the incorporation of unsaturated C18 fatty acids into triacyglycerol (TAG). This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DGAT3-mediated TAG biosynthesis pathway.  相似文献   

17.
The insect fat body and the adipose tissue of vertebrates store fatty acids (FA) as triacylglycerols (TG). However, the fat body of most insects has the unique ability to rapidly produce and secrete large amounts of diacylglycerol (DG). Monoacylglycerol acyltransferase (MGAT), which catalyzes the synthesis of DG from MG, and a diacylglycerol acyltransferase (DGAT), which catalyzes the synthesis of TG from DG, are key enzymes in the metabolism of neutral glycerides. However, very little is known about these acyltransferases in insects. In the present study we have cloned two predicted MGATs and a DGAT from Manduca sexta and compared their sequences with predicted MGAT and DGAT homologs from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1. The apparent absence of a DGAT2 gene in insects would represent a major difference with vertebrates, which contain DGAT1 and DGAT2 genes. Insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. A number of conserved phosphorylation sites of potential physiological significance were identified among insect proteins and among insect and vertebrate proteins. DGAT1 and MGAT are expressed in fat body, midgut and ovaries. The relative rates of utilization of FAs for the synthesis of DG and TG correlated with the relative expression levels of MGAT and DGAT suggesting that regulation of the expression levels of these acyltransferases could be determining whether the fat body secretes DG or stores fatty acids as TG. The expression patterns of the acyltransferases suggest a role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.  相似文献   

18.
Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.  相似文献   

19.
The objectives of the present study were focused on detecting deletion mutation in bovine AMPD1 gene, and analyzing its effect on body measurement and carcass traits in Qinchuan cattle by using DNA sequencing and agarose electrophoresis methods. The 198-bp PCR products of AMPD1 gene exhibited three genotypes and two alleles were revealed: A and B. The frequencies of genotype AA/AB/BB in Qinchuan populations was 0.7163, 0.2233 and 0.0605. The χ2-test analysis demonstrated that the breed was not in agreement with Hardy–Weinberg equilibrium (P < 0.05). The association of the 18-bp deletion mutation of AMPD1 gene with body measurement and carcass traits of Qinchuan cattle were analyzed. The cattle with AA genotype had slaughter weight and carcass weight than those with genotype AB (P < 0.01 or P < 0.05). These results suggest that the 18-bp deletion mutation may influence the carcass traits in Qinchuan cattle.  相似文献   

20.
Nawabi P  Lykidis A  Ji D  Haldar K 《Eukaryotic cell》2003,2(5):1128-1131
Here we show that blood-stage Plasmodium falciparum organisms accumulate a high mass of triacylglycerol and diacylglycerol. However, we failed to detect cholesterol esters, a second neutral lipid species reported to be important for a related apicomplexan, Toxoplasma gondii. Evidence for P. falciparum and T. gondii homologues of acyl coenzyme A:diacylglycerol acyltransferase suggests that acylglycerols may be the conserved neutral lipids in apicomplexans.  相似文献   

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