Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

FLJ14813 missense mutation: a candidate for autosomal dominant thrombocytopenia on human chromosome 10

The gene for a novel nonsyndromic autosomal dominant thrombocytopenia has been previously mapped to a region on human chromosome 10p11-12 (THC2, OMIM number *188000). This disorder is characterized by moderate thrombocytopenia and incomplete differentiation of megakaryocytes. We report here a novel missense mutation in the human gene FLJ14813 that segregates perfectly with thrombocytopenia in our kindred of 51 family members. The mutation is not detected in 94 random unrelated and unaffected individuals, nor is it reported in the Entrez single nucleotide polymorphism (SNP) database. A substitution of cytosine for guanidine (G to C) at nucleotide position 565 was present in all thrombocytopenic family members, causing a predicted substitution of aspartic acid for glutamic acid (E167D) in exon four.     

牛FSHR基因第10外显子单核苷酸多态性及其与双胎性状的关系

以秦川牛和荷斯坦奶牛的双胎母牛和单胎母牛为实验材料 ,以牛的FSHR基因的第 10个外显子作为标记牛双胎性状的候选基因 ,用SNP法进行了多态检测 .结果发现 ,在秦川牛的双胎母牛中突变率 6 0 % (6 10 ) ,而在单胎母牛中突变率为 2 0 % (2 10 ) ;在荷斯坦奶牛中 ,双胎母牛突变率为31 2 5 % (5 16 ) ,单胎母牛突变率为 6 6 7% (1 15 ) ;由此可见双胎牛和单胎牛二者之间FSHR基因的第 10个外显子的突变率差异明显 .这表明 ,选择FSHR基因的第 10个外显子有可能作为双胎性状的候选基因 .序列分析发现 ,在FSHR基因的第 15 0 6位碱基发生了突变 (T→C) ,但氨基酸没有发生变化 .     

四引物扩增受阻突变体系PCR快速测定绵羊 BMPR-IB基因型方法的建立

四引物ARMS PCR是检测SNP有效、快速、简便的方法.绵羊BMPR-lB基因是控制Booroola绵羊多胎性状的主效基因,此研究目的在于建立一种对BMPR-IB基因四引物ARMS PCR检测方法.根据四引物ARMS PCR技术原理,在绵羊BMPR-IB基因突变位点(A746G)设计一对特异性引物,并在突变点两侧设计一对参照引物,用来扩增含有突变点的DNA片段,可在一步PCR反应中根据电泳图谱准确判断绵羊个体的BMPR-IB基因型,对比PCR-RFLP检测结果表明,所建立的方法简单,操作简便,大大提高了检测效率.     

Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp

In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.     

A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme. Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity.     

Variation and expression of KAP9.2 gene affecting cashmere trait in goats

Keratin-associated proteins 9.2 (KAP9.2) gene encodes one of the ultra high sulfur KAPs. Variation in KAP genes may affect the structure of KAPs and hence cashmere characteristics. In order to test the association between the polymorphism of KAP9.2 gene and cashmere trait, DNA sequencing was used to detect a novel C/T polymorphism of KAP9.2 gene from a genomic DNA pool. The mutation could be recognized by Pst I restriction enzyme. To Shanbei white cashmere goat, Inner Mongolia white cashmere goat and Guanzhong dairy goat, the genotypic frequencies of TT, TC and CC from total 1,236 animals were as follows: 0.047, 0.519 and 0.434; 0.180, 0.592 and 0.228; 0.431, 0.544 and 0.025. The allelic frequencies of T and C were 0.307 and 0.693; 0.476 and 0.524; 0.703 and 0.297, respectively, in breeds mentioned above. The frequency of C allele between cashmere and dairy goat was significant (P?<?0.01). To provide support for the hypothesis that SNP 586 was responsible for KAP9.2 expression, quantitative real-time PCR analysis revealed that the expression level of KAP9.2 was reduced in individuals bearing genotype CC compared with TT individuals, suggesting that C was the nucleotide causing decreased expression of KAP9.2 or was in linkage disequilibrium with the causative SNP. The 586C/T SNP found in this study might control translation or stability of KAP9.2 mRNA, which would be beneficial for marker assistant selection in cashmere goat breeding.     

Characterization of the quantitative trait locus for haloperidol-induced catalepsy on distal mouse chromosome 1

We report here the confirmation of the quantitative trait locus for haloperidol-induced catalepsy on distal chromosome (Chr) 1. We determined that this quantitative trait locus was captured in the B6.D2- Mtv7a /Ty congenic mouse strain, whose introgressed genomic interval extends from approximately 169.1 to 191.3 Mb. We then constructed a group of overlapping interval-specific congenic strains to further break up the interval and remapped the locus between 177.5 and 183.4 Mb. We next queried single nucleotide polymorphism (SNP) data sets and identified three genes with nonsynonymous coding SNPs in the quantitative trait locus. We also queried two brain gene expression data sets and found five known genes in this 5.9-Mb interval that are differentially expressed in both whole brain and striatum. Three of the candidate quantitative trait genes were differentially expressed using quantitative real-time polymerase chain reaction analyses. Overall, the current study illustrates how multiple approaches, including congenic fine mapping, SNP analysis and microarray gene expression screens, can be integrated both to reduce the quantitative trait locus interval significantly and to detect promising candidate quantitative trait genes.     

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last steo of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for detects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA is cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.     

Capn10, a candidate gene responsible for type 2 diabetes mellitus in the OLETF rat

The rat strain Otsuka Long-Evans Tokushima Fatty (OLETF) is an animal model for type 2 diabetes mellitus. Nidd8/of has been identified as one of 14 quantitative trait loci (QTLs) involved in the diabetes by a whole genome search in 160 F2 progenies obtained by mating the OLETF and F344 rats. Comparative mapping between human and rat indicated that the Nidd8/of genomic region, near D9rat21 on rat chromosome 9, contains the calpain10 (Capn10) gene, which is putative type 2 diabetes-susceptibility gene in humans. In this study, we found no difference in Capn10 mRNA expression in the heart, liver, skeletal muscle and pancreas between OLETF and F344 rats at 5 and 10 weeks of age. However, we found a single nucleotide polymorphism (SNP) (A/A genotype in OLETF and G/G genotype in F344 and LETO rats) at the base 583 downstream from the translation start site in the rat Capn10 cDNA sequence. This SNP was deduced to substitute serine (OLETF) for glycine (F344 and LETO) at the 195 amino acid residue within the protease domain of rat Capn10. Because serine is generally not interchangeable with glycine in respect of the protein structure and function, it was deduced that the A/A genotype in OLETF is not a 'safe' mutation. This non-conservative amino acid substitution might be associated with susceptibility to type 2 diabetes in OLETF rats.     

A grapevine (Vitis vinifera L.) deoxy-d-xylulose synthase gene colocates with a major quantitative trait loci for terpenol content

Linalool, geraniol, nerol, citronellol and α-terpineol are isoprenoid molecules responsible for specific aromas found in grapes and wines. Total concentrations (free and bound forms) of these compounds were measured in the skins of mature berries during 2 successive years in two progenies obtained from Muscat Ottonel and Gewurztraminer selfings. Partial genetic maps based on microsatellite markers were constructed and several quantitative trait loci (QTLs) related to terpenol content were detected. A major QTL on linkage group (LG) 5 colocated with a deoxy-d-xylulose synthase gene, coding for the first enzyme of the plastidial isoprenoid biosynthesis pathway. The number of favourable alleles at this locus determined the level of terpenol synthesis. A second QTL, on LG 10, was found to determine the balance linalool versus geraniol and nerol in the Muscat self-progeny plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

A missense mutation (c.1963A<G) of the complementary component 2 (C2) gene is associated with serum Ca++ concentrations in pigs

Serum Ca(++) levels play important roles in the humoral immunity. The aim of this study was to detect quantitative trait loci and the associated positional candidate genes affecting baseline serum Ca(++) concentrations. A genome-wide association study was conducted in an F(2) intercross population between Landrace and Korean native pigs using the porcine single nucleotide polymorphism (SNP) 60?K beadchip and the PLINK program based on linear regression. Data used in the study included 410 F(2) pigs. All experimental animals were genotyped with 36,613 SNP markers located throughout the pig autosomes. We identified a strong association between a SNP marker on chromosome 7 and serum Ca(++) levels (DIAS0002191, genomic control-corrected P?=?7.7?×?10(-5)). The position of DIAS0002191 was closely located to SLA class III region containing the C2 gene encoding the complementary component 2 protein, a protein which is important in the humoral immune responses. De novo sequencing of the porcine C2 gene revealed a missense mutation [c.1963A相似文献   

Molecular characterization, polymorphism of the ACOX1 gene and association with ultrasound traits in Bos taurus

Acyl-coenzyme A oxidase 1 (ACOX1) is the first enzyme in peroxisomal fatty acid β-oxidation; it is rate-limiting and plays a key role in fatty acid metabolism and fat deposition. ACOX1 is an important candidate gene for meat quality selection through marker-assisted selection. Genomic structural analysis showed that bovine ACOX1 shares 86% identity with human ACOX1. Using PCR-SSCP technology, we discovered a single nucleotide polymorphism (SNP) (A1865C) in exon 13 of the ACOX1 gene. Allele frequencies of this SNP were investigated and evaluated with the χ(2) test in 641 cattle populations; only the Jiaxian red population was not in Hardy-Weinberg equilibrium. Gene heterozygosity, effective allele numbers and polymorphism information content of the bovine ACOX1 locus in seven populations varied from 0.2778 to 0.4954, 1.3846 to 1.9817 and 0.2392 to 0.3727, respectively. We also looked for a potential association of this SNP with ultrasound traits in 327 individuals and found a significant effect on ultrasound backfat thickness and ultrasound marbling score (P < 0.05). Meat quality traits were analyzed in another 71 Qinchuan individuals to determine associations with genotype. Animals with genotype AA had higher mean values of backfat thickness than those with genotypes AC and CC. A represents the base before mutation and C represents the base after mutation. We conclude that this SNP of the ACOX1 gene has potential as a genetic marker for meat quality traits in cattle reproduction and breeding.     

Origin of agouti-melanistic polymorphism in Wild Black Rats (Rattus rattus) inferred from Mc1r gene sequences

We examined nucleotide changes that underlie coat color variation in Black Rats (the Rattus rattus species complex), which show polymorphism in dorsal fur color, including either grayish brown (agouti) or black (melanistic) forms. We examined the full coding sequence of a gene known to produce melanism in other vertebrates-melanocortin-1-receptor gene Mc1r (954 bp) -using samples of both R. rattus (with 2n = 38) and its close relative Asian Black Rat (R. tanezumi; 2n = 42). We used 61 specimens from Japan with karyotype-known individuals and four samples from Pakistan. We found 11 allele sequences and constructed a network tree that shows two distinct clusters, with allelic segregation according to karyotype and by inference, representing the two species. We found that a nucleotide substitution from G to A at site 280, producing an amino acid change from glutamic acid to lysine, was associated with the dominant trait of the melanistic form of the coat color in R. rattus. Notably, the derived SNP 280A was found in a single allele, with the ancestral SNP 280G present in seven alleles. By contrast, all three alleles for R. tanezumi retain the ancestral SNP 280G. These results suggest a possible recent origin of melanism in R. rattus.     

Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes.

The protein composition of the grape (Vitis vinifera cv Muscat of Alexandria) berry was examined from flowering to ripeness by gel electrophoresis. A protein with an apparent molecular mass of 24 kD, which was one of the most abundant proteins in extracts of mature berries, was purified and identified by amino acid sequence to be a thaumatin-like protein. Combined cDNA sequence analysis and electrospray mass spectrometry revealed that this protein, VVTL1 (for V. vinifera thaumatin-like protein 1), is synthesized with a transient signal peptide as seen for apoplastic preproteins. Apart from the removal of the targeting signal and the formation of eight disulfide bonds, VVTL1 undergoes no other posttranslational modification. Southern, northern, and western analyses revealed that VVTL1 is found in the berry only and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening. The exact role of VVTL1 is unknown, but the timing of its accumulation correlates with the inability of the fungal pathogen powdery mildew (Uncinula necator) to initiate new infections of the berry. Western analysis revealed that the presence of thaumatin-like proteins in ripening fruit might be a widespread phenomenon.