Molecular Cloning and Expression Analysis of Transformer‐2 Gene During Development in Macrobrachium nipponense (de Haan 1849)

The transformer‐2 gene (tra‐2) plays a key role in the sexual differentiation regulatory hierarchy. In this study, tra‐2 gene homologs designated as Mntra‐2 was cloned and characterized from Macrobrachium nipponense. The full‐length cDNA of Mntra‐2 consists of 1724 bp with an open reading frame (ORF) encoding 192 amino acids, an 827 bp 5′‐untranslated region (UTR) and a 318 bp 3′‐UTR. The predicated molecular mass of Mntra‐2 was 20.805 kDa with an estimated theoretical isoelectric point of 10.36. The deduced amino acid sequence shares high homology with Penaeus monodon. Real‐time quantitative polymerase chain reaction (RT‐qPCR) analyses demonstrated that the expression levels of Mntra‐2 varied significantly during different developmental stages of embryogenesis, larvae, and post‐larvae and in various adult tissues. During embryogenesis, the expression level of Mntra‐2 was slightly higher at the cleavage stage than at the blastula stage, and reached the highest level at the nauplius stage. During the larvae, the Mntra‐2 expression gradually increased from 1 d larvae post hatch (L1) to L10 and decreased to a lowest level at the end of metamorphosis. During the post‐larvae, the Mntra‐2 expression was higher level at the 5 d after the metamorphosis (P5). RT‐qPCR showed the Mntra‐2 mRNA was expressed in ovary, testis, muscle, heart, abdominal ganglion, brain, and intestine with the highest level of expression in muscle and intestine. The results indicate that Mntra‐2 is an arthropods tra‐2 homolog and probably plays important roles in embryonic development and sex differentiation of M. nipponense .     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

淇河鲫cyp19a1b基因的克隆表达及芳香化酶抑制剂对其表达的影响

为探索脑型芳香化酶基因(cyp19a1b)在雌核发育三倍体鱼淇河鲫性别决定与分化过程中的作用,利用RACE方法克隆淇河鲫cyp19a1b基因cDNA全长序列,采用Real-time PCR分析其在不同组织、胚胎及胚后不同发育时期的表达情况,同时检测芳香化酶抑制剂Letrozole诱导性逆转及腹腔注射人绒毛膜促性腺激素(hCG)后cyp19a1b在脑中的表达情况。结果显示,淇河鲫cyp19a1b cDNA全长2 984 bp(GenBank ID:MF926270),包含132 bp5′非编码区,1 319 bp 3′非编码区,1 533 bp开放读码框,编码510个氨基酸残基;氨基酸序列比对及系统进化分析结果显示,淇河鲫Cyp19a1b与其他鲤科鱼类同源性较高,与哺乳类、爬行类等脊椎动物同源性较低,这与其分类地位一致;组织分布检测结果显示,cyp19a1b基因在淇河鲫脑中表达量最高,在卵巢等其他组织中表达量较低;Realtime PCR结果表明,在胚胎发育过程中cyp19a1b在外源精子刺激后从囊胚期开始上调,神经胚期达到最高,随后降低;出膜后随着发育的进行,该基因在脑中表达量逐步上调,在躯体中一直维持在较低水平;除此之外,伴随着Letrozole诱导的性逆转,cyp19a1b在脑中表达量降低;腹腔注射hCG可以促进cyp19a1b在脑中表达。研究表明,淇河鲫cyp19a1b基因可能通过参与神经系统的形成及神经内分泌活动,在性别决定与分化过程中起到一定的作用。     

Molecular characterization and identification of facilitative glucose transporter 2 (GLUT2) and its expression and of the related glycometabolism enzymes in response to different starch levels in blunt snout bream (<Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>)

Facilitative glucose transporters (GLUT) are transmembrane transporters involved in glucose transport across the plasma membrane. In this study, blunt snout bream GLUT2 gene was cloned, and its expression in various tissues and in liver in response to diets with different carbohydrate levels (17.1; 21.8; 26.4; 32.0; 36.3; and 41.9% of dry matter). Blunt snout bream GLUT2 was also characterized. A full-length cDNA fragment of 2577 bp was cloned, which contains a 5′-untranslated region (UTR) of 73 bp, a 3′-UTR of 992 bp, and an open reading frame of 1512 bp that encodes a polypeptide of 503 amino acids with predicted molecular mass of 55.046 kDa and theoretical isoelectric point was 7.52. The predicted GLUT2 protein has 12 transmembrane domains between amino acid residues at 7–29; 71–93; 106–123; 133–155; 168–190; 195–217; 282–301; 316–338; 345–367; 377–399; 412–434; and 438–460. Besides, the conservative structure domains located at 12–477 amino acids belong to the sugar porter family which is the major facilitator superfamily (MFS) of transporters. Blunt snout bream GLUT2 had the high degree of sequence identity to four GLUT2s from zebrafish, chicken, human, and mouse, with 91, 63, 57, and 54% identity, respectively. Quantitative real-time (qRT) PCR assays revealed that GLUT2 expression was high in the liver, intestine, and kidney; highest in the liver and was regulated by carbohydrate intake. Compared with the control group (17.1%), fed by 3 h with higher starch levels (32.0; 36.3; and 41.9%), increased plasma glucose levels and glycemic level went back to basal by 24 h after treatment. Furthermore, higher dietary starch levels significantly increase GLUT2, glucokinase (GK), and pyruvate kinase (PK) expression and concurrently decrease phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) mRNA levels (P?<?0.05), and these changes were also back to basal levels after 24 h of any dietary treatment. These results indicate that the blunt snout bream is able to regulate their ability to metabolize glucose by improving GLUT2, GK, and PK expression levels and decreasing PEPCK and G6P expression levels.