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瓦氏黄颡鱼生长激素基因克隆及其组织特异性表达分析 总被引：1，自引：1，他引：0
生长激素(growth hormone,GH)对脊椎动物的生长发育及代谢具有重要作用。采用RT-PCR和RACE技术,克隆了瓦氏黄颡鱼垂体GH cDNA全长序列,应用real-time qPCR法对不同组织中GH mRNA的表达进行检测。序列分析表明,GH cDNA(GenBank登录号:GU395549)序列全长1 203 bp,其5’端非编码区77 bp、3’端非编码区523 bp,开放阅读框(open reading frame,ORF)603 bp,由此推导GH前体蛋白由200个氨基酸组成。同源性比较结果表明,瓦氏黄颡鱼与同目鱼类的GH编码序列同源性较高,与哺乳类和鸟类的同源性较低。Real-time qPCR结果显示,GH mRNA在垂体中的表达量最高,其次是脑、肌肉、肝脏、脂肪组织、胃、脾脏、卵巢或精巢,而在肾脏、心脏、鳃和肠中没有明显的表达。实验结果表明,GH基因在瓦氏黄颡鱼组织中广泛表达,提示GH可能以旁分泌或自分泌的方式对其生长和繁殖发挥重要作用。 相似文献
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Ghrelin was recently demonstrated as an endogenous ligand of the growth hormone (GH) secretagogue receptor (GHS-R), which could promote the release of GH in mammal significantly. The present study conducted to determine whether ghrelin stimulate the release and synthesis of GH in orange-spotted grouper (Epinephelus coioides). Rat ghrelin was incubated with the pituitary fragments of grouper in static culture system. The culture medium was collected at 1, 6, 12, 18 and 24 h after incubation to detect the contents of GH by homologous radioimmunoassay. The level of GH mRNA in the pituitary fragments was measured by a sensitive chemiluminescent ribonuclease protection assay. The results showed that rat ghrelin not only stimulated the release of GH but also augmented the GH mRNA level in grouper. It suggested that the ghrelin-like peptide and the GHS-R involved in the regulation of GH synthesis and release in grouper. The present study would provide a better understanding of the regulatory mechanism of GH release in marine fish. 相似文献
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The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned
from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that
it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin
than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene
in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels
in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low
level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated
that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore,
caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues.
Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on
the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an
internal control for RT-PCR analysis of MT effects on gene expression in these tissues. 相似文献
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Yun Li Xiaochun Liu Yong Zhang Xilan Ma Haoran Lin 《Fish physiology and biochemistry》2013,39(3):605-613
Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3 mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4 mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8 weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1–3 mg/g diet) is an effective, economical and feasible feed additive in orange-spotted grouper culture. 相似文献
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斜带石斑白细胞cDNA文库的构建 总被引：5，自引：2，他引：5
用Tripure试剂盒提取斜带石斑白细胞总RNA,反转录合成第一链cDNA,进行长距离PCR,蛋白酶K消化,Sfi I酶切,通过CHROMASPIN-400柱,回收大于500bp的cDNA,与λTripIEx2载体连接,体外包装后构建了斜带石斑白细胞的cDNA文库.库容1.65×1.06,重组频率82%,扩增后滴度7.5×109pfu·mL-1.插入片断长度500～2300bp,最多的是在750～1000bp范围.本文库可作为筛选斜带石斑细胞因子基因的重要资源. 相似文献
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The role of growth hormone (GH) in regulating hepatic mRNA expression of insulin-like growth factor-I (IGF-I) and IGF binding
proteins (IGFBPs) in yellowtail Seriola quinqueradiata was examined using in vivo and in vitro assays. Yellowtail hepatic IGF-I, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-5 mRNAs were
measured by real-time quantitative RT-PCR. Intraperitoneal injection of recombinant GH of chum salmon Oncorhynchus keta (rsGH) at a dose of 1 μg/g body weight resulted in a significant increases in hepatic IGF-I, IGFBP-3, and IGFBP-5 mRNA levels,
whereas significant reductions in hepatic IGFBP-1 and IGFBP-2 mRNA levels were observed. For in vitro assays, liver slices
were incubated with rsGH at different concentrations (doses: 0, 1, 10, 100, 500, and 1,000 ng/ml). Liver slices incubated
with 100 ng/ml rsGH elicited a significant increase in IGF-I mRNA level. Similarly, a slight increase in IGFBP-3 and IGFBP-5
mRNA levels were also observed in liver explants incubated with rsGH. In contrast, a significant decline in IGFBP-1 mRNA levels
was observed in liver slices incubated with 1,000 ng/ml rsGH. A slight decline in the level of IGFBP-2 mRNA was noted in liver
explants with rsGH treatment. This study demonstrates the modulating effect of GH on the IGF system. 相似文献
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Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) 
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下载免费PDF全文 Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper. 相似文献
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T. T. Chen L. B. Agellon C. M. Lin H. J. Tsai Peijun Zhang L. I. González-Villasénor D. A. Powers 《Fish physiology and biochemistry》1989,7(1-6):381-385
The primary structures of two rainbow trout growth hormone mRNAs (GH1 and GH2) have been deduced by direct sequencing of their
respective cDNA clones and portions of the mRNA. Both GH1 and GH2 mRNA contain open reading frames comprised of 630 nucleotides
and encode 210 amino acid residues of which 11 are variant. The translated regions of both mRNA are flanked by a short but
rather conserved 5′-end, and a relatively long but highly diverged 3′-end. The differences at translated and 3′-untranslated
regions suggest that the GH1 and GH2 mRNA originate from different loci. The GH1 and GH2 mRNA are likely transcribed from
two distinct loci which were duplicated during tetraploidization of salmonid genome between 50 to 100 million years ago.
The GH2 gene has been isolated and sequenced from a rainbow trout genomic library. This gene spans a region of approximately
4 kilobases. The trout GH gene is comprised of 6 exons and 5 introns, in contrast to 5 exons and 4 introns in mammals. The
additional intron in the trout gene interrupts the translated regions that are analogous to the last exon of the mammalian
counterpart. The alleged internally repeating sequences in mammalian GH, prolactin (Pr1) and placental lactogen (PL) are not
observed in the predicted polypeptide sequence of trout GH. In addition, direct repeats that flank exons I, III and V of mammalian
GH, Pr1 and PL genes are absent in trout gene. These findings indicate that the rainbow trout GH gene structure does not support
the current hypothesis that internally repeated regions in GH, Pr1 and PL arose from a small primordial gene. 相似文献
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Insulin-like growth factors I and II (IGF-I and IGF-II) are two highly homologous mitogenic peptides that are expressed ubiquitously and show diverse effects on development, growth, and metabolism. The cDNA encoding IGF-I of a teleost, the orange spotted grouper (Epinephelus coioides) was produced from liver by RT-PCR, and rapid amplification of cDNA ends, RACE. Typically, the deduced 186 amino acid protein contains a signal peptide, B, C, A, D and E domains. On the amino acid level, grouper IGF-I shares 97.3% similarity with black seabream (Sparus macrocephalus) with the differences focusing on the B and C domains. The analysis of the E domain showed that grouper IGF-I belonged to Ea-4 type. When mature amino acid sequence was compared with other vertebrates, it revealed higher similarity with black seabream and halibut, while lower similarity with human and mouse. The expression of IGF-I mRNA in adult tissues was studied using RT-PCR. IGF-I mRNA expression level in the liver was significantly higher than those in the brain and muscles. In other tissues, low amount of IGF-I mRNA expression was also detected. The coding region of IGF-I cDNA for mature IGF-I protein was subcloned into an expression plasmid pTRX and fused with E. coli thioredoxin (Trx). Moreover, we have successfully developed an expression system in E. coli to overproduce recombinant grouper IGF-I. Using western blotting, we found that the fusion protein could blot with antiserum to barramundi IGF-I further confirming the immunoactivity of the recombinant IGF-I. 相似文献
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Molecular cloning and functional characterization of a putative Elovl4 gene and its expression in response to dietary fatty acid profiles in orange‐spotted grouper Epinephelus coioides 下载免费PDF全文
下载免费PDF全文 Songlin Li Óscar Monroig Juan Carlos Navarro Yuhui Yuan Wei Xu Kangsen Mai Douglas R Tocher Qinghui Ai 《Aquaculture Research》2017,48(2):537-552
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Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate 下载免费PDF全文
下载免费PDF全文 Satoshi Kubota Amphai Longloy Arkom Singhabun Wanpen Khammee Kanonkporn Kessuwan Paiboon Bunlipatanon Akiyuki Ozaki Kom Silapajarn Varin Tanasomwang Nobuaki Okamoto Takashi Sakamoto 《Aquaculture Research》2017,48(12):5913-5927
Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F1 hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species. 相似文献
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Xiao Hui Li Jun Jie Bai Xing Ye Yin Chang Hu Sheng Jie Li Ling Yun Yu 《Fisheries Science》2009,75(2):351-358