Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5′/3′-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24 h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole (Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males (P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.     

The co-existence of two growth hormone receptors and their differential expression profiles between female and male tongue sole (Cynoglossus semilaevis)

Growth hormone receptor (Ghr) is a single-transmembrane pass protein which is important in initiating the ability of growth hormone (Gh) to regulate development and somatic growth in vertebrates. In this study, molecular cloning, expression analysis of two different ghr genes (ghr1 and ghr2) in the tongue sole (Cynoglossus semilaevis) was conducted. As a result, the ghr1 and ghr2 cDNA sequences are 2364 bp and 3125 bp, each of which encodes a transmembrane protein of 633 and 561 amino acids (aa), respectively. Besides, the ghr1 gene includes nine exons and eight introns. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. Both the ghr1 and ghr2 were predominantly expressed in the liver, and the ghr1 expression level in normal males was 1.6 and 1.4 times as much as those in females and extra-large males, while the ghr2 mRNA expression level in normal males was 1.1 and 1.2 times as much as those in females and extra-large males, respectively. Ontogenetic expression analysis at early life stages indicated that the ghr1 and ghr2 mRNAs were detected at all of the 35 sampling points (from oosphere to 410 days-old). Furthermore, the sex differences in ghr mRNA expressions were also examined by using a full-sib family of C. semilaevis. Significantly higher levels of ghr1 mRNA were observed in males than in females at most stages of the sampling period (P < 0.01). The ghr2 mRNA expression at most stages exhibited a significant sexual difference at each sampling point (P < 0.01) without any variation trend related with the sexes during the whole sampling period.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262 bp in length and has an open reading frame (ORF) of 1038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.     

Molecular Characterization and Expression Analysis of VSIG4 from the Asian Yellow Pond Turtle, Mauremys mutica

V-set and immunoglobulin domain-containing protein 4 (VSIG4), a member of the immunoglobulin superfamily, plays an important role in the immune system. This study isolated and characterized a cDNA encoding VSIG4 (MaVSIG4) from the Asian yellow pond turtle (Mauremys mutica). The MaVSIG4 cDNA is 1840 bp long and contains an open reading frame of 1,182 bp that encodes a polypeptide of 372 amino acids. The genomic sequence of MaVSIG4 spans 7,682?bp, with six exons and five introns. The phylogenetic tree shows that MaVSIG4 is most closely related to Gallus gallus VSIG4. The expression analysis by real-time PCR reveals that MaVSIG4 is ubiquitously expressed in various healthy tissues, with a higher expression level in the liver. After immune stimulation, the expression level of MaVSIG4 sharply decreased in the liver, heart, and kidney at 12?h (P?<?0.01). These results provide a basis for further study of the function of MaVSIG4 in the turtle’s immune system.     

cDNA,genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.