Genome-wide analysis of chloride channel-encoding gene family members and identification of CLC genes that respond to Cl-/salt stress in upland cotton.

Chloride channels (CLCs) are kinds of anion transport protein family members that are mainly distributed in cell endomembrane systems of prokaryotic and eukaryotic organisms and mediate anion (Cl-, as a representative) transport and homeostasis. Some CLC genes have been reported to be involved in Cl-/salt tolerance of plants exposed to NaCl stress. Through BLAST in cotton database, a total of 22 CLCs were identified in genomes A and D in upland cotton (Gossypium hirsutum L.), and except for GhCLC6 and GhCLC17, they formed highly similar homologous genes pairs. According to the prediction in PlantCARE database, many cis-acting elements related to abiotic stress responses, including ABREs, AREs, GT-1s, G-boxes, MYBs, MYCs, etc., were found in the promoters of GhCLCs. qRT-PCR revealed that most GhCLC gene expression was upregulated in the roots and leaves of cotton seedlings under salt stress, and those of homologous GhCLC4/15, GhCLC5/16, and GhCLC7/18 displayed more obvious expression. Furthermore, according to leaf virus-induced gene silencing (VIGS) assay and compared with the salt-stressed GhCLC4/15- and GhCLC7/18-silenced cotton plants, the salt-stressed GhCLC5/16-silenced plants displayed relatively better growth with significant increases in both Cl- content and Cl-/NO3- ratio in the roots and drop of the same parameters in the leaves. These results indicate that homologous GhCLC5/16, with the highest NaCl-induced upregulation of expression and the maximum number of MYC cis-acting elements, might be the key members contributing to cotton Cl-/salt tolerance by regulating the transport, interaction and homeostasis of Cl- and NO3-.

Two myostatin genes exhibit divergent and conserved functions in grass carp (Ctenopharyngodon idellus).

Myostatin (MSTN) is an important negative regulator of myogenesis, which inhibits myoblast proliferation and differentiation. Here, we report the isolation and characterization of two mstn genes in grass carp (Ctenopharyngodon idellus). Grass carp mstn-1 and mstn-2 cDNAs are highly divergent, sharing a relatively low amino acid sequence identity of 66%. In adult fish, both orthologs are expressed in numerous tissues and they are differentially regulated during a fasting/refeeding treatments. During embryogenesis, the mRNA levels of both mstn-1 and -2 were upregulated significantly at the beginning of somitogenesis, and maintained at high levels until hatching. Using in situ hybridization, grass carp mstn-1 mRNA was found to ubiquitously express at 12hpf, with strong signals in the notochord, and in the eyes, brain and tailbud at 24hpf, and in brain and notochord at 36hpf. In comparison, the mstn-2 mRNA can be detected in the eyes, brain and notochord at 24hpf, and in the notochord and hindbrain at 36hpf. Further overexpression of mstn-1 mRNA caused a strongly ventralized phenotype by inhibiting dorsal tissue development, while injection of mstn-2 mRNA resulted in obvious embryonic abnormalities in grass carp. These results provide some new insights into the functional conservation and divergence of mstn genes in teleost species.

Functional conservation and divergence of duplicated insulin-like growth factor 2 genes in grass carp (Ctenopharyngodon idellus).

Insulin-like growth factor 2 (IGF2) is a potent mitogenic and survival factor involved in the regulation of growth, development and reproduction in animals. Only one IGF2 gene exists in mammals. Recently, two igf2 genes have been identified in zebrafish, which presumably resulted from gene duplication. However, sequence information of duplicated igf2s and their functional regulation in other teleost fish is still unknown. Here, we report the identification of two igf2 cDNAs in grass carp, Ctenopharyngodon idellus. Like their human ortholog, grass carp igf2a and igf2b mRNAs encoded two structurally distinct mature IGF peptides. Both of them were detected by RT-PCR throughout embryogenesis. Ubiquitous expression of igf2b mRNAs was observed in embryos, whereas igf2a mRNAs were expressed mainly in the notochord and brain with in situ hybridization. In adult fish, igf2b mRNAs were transcribed in multiple tissues, whereas igf2a mRNAs were detected mainly in the liver. Hepatic levels of igf2a and igf2b transcripts were both up-regulated by growth hormone injection. Furthermore, the levels of hepatic igf2a and igf2b mRNAs decreased significantly during starvation and were rebounded rapidly after re-feeding. Our results suggest that duplicated igf2 genes have evolved divergent yet played an overlapping biological role in regulating grass carp growth and development.

HIF-1alpha and -2alpha genes in a hypoxia-sensitive teleost species Megalobrama amblycephala: cDNA cloning, expression and different responses to hypoxia.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF) alpha subunits, i.e. HIF-1alpha, -2alpha and -3alpha. In fish, the molecular constructions, expression characteristics and hypoxic regulation of HIF-alpha subunits are still not well known. In this study, we identified the HIF-1alpha and HIF-2alpha full-length cDNAs in a hypoxia-sensitive fish species Wuchang bream, Megalobrama amblycephala. The whole length of HIF-1alpha cDNA was 3,815bp, consisting of an open reading frame (ORF) encoding 774 amino acid (aa) residues. The HIF-2alpha cDNA totaled 3,121-bp including an 835-aa ORF. The Wuchang bream HIF-1alpha and HIF-2alpha subunits were structurally similar in the DNA-binding and dimerization domains, but differed in the transactivation domain. In adult fish, both HIF-1alpha and HIF-2alpha mRNAs were detected in different tissues under normoxic conditions. HIF-1alpha mRNA was highly expressed in the liver, gill and testis, whereas HIF-2alpha mRNA was abundantly expressed in most of the Wuchang bream tissues. Both HIF-1alpha and HIF-2alpha mRNAs were detected in all stages of embryogenesis and expressed in a ubiquitous pattern. In contrast to HIF-1alpha, the mRNA levels of HIF-2alpha fluctuated in different stages, with higher expression in the zygote, 8-, 28-, 48- and 52-hr post fertilization (hpf) embryos. During hypoxic treatment, the mRNA levels of HIF-2alpha were significantly (p<0.01) up-regulated to 910% in the liver and 320% in the kidney, whereas no significant changes of HIF-1alpha mRNA were observed in the corresponding tissues. These results suggest that the Wuchang bream HIF-1alpha and -2alpha would be involved in different physiological functions under normoxia and hypoxia situations.