Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

Comparative transcriptome analysis provides insight into regulation pathways and temporal and spatial expression characteristics of grapevine (Vitis vinifera) dormant buds in different nodes.

BACKGROUND: Bud dormancy is a strategic mechanism plants developed as an adaptation to unfavorable environments. The grapevine (Vitis vinifera) is one of the most ancient fruit vine species and vines are planted all over the world due to their great economic benefits. To better understand the molecular mechanisms underlying bud dormancy between adjacent months, the transcriptomes of 'Rosario Bianco' grape buds of 6 months and three nodes were analyzed using RNA-sequencing technology and pair-wise comparison. From November to April of the following year, pairwise comparisons were conducted between adjacent months. RESULTS: A total of 11,647 differentially expressed genes (DEGs) were obtained from five comparisons. According to the results of cluster analysis of the DEG profiles and the climatic status of the sampling period, the 6 months were divided into three key processes (November to January, January to March, and March to April). Pair-wise comparisons of DEG profiles of adjacent months and three main dormancy processes showed that the whole grapevine bud dormancy period was mainly regulated by the antioxidant system, secondary metabolism, cell cycle and division, cell wall metabolism, and carbohydrates metabolism. Additionally, several DEGs, such as VvGA2OX6 and VvSS3, showed temporally and spatially differential expression patterns, which normalized to a similar trend during or before April. CONCLUSION: Considering these results, the molecular mechanisms underlying bud dormancy in the grapevine can be hypothesized, which lays the foundation for further research.

Identification and Characterization of the DMRT11E Gene in the Oriental River Prawn Macrobrachium nipponense.

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.

Genome-wide identification, evolution of histone lysine demethylases (KDM) genes and their expression during gonadal development in Nile tilapia.

Histone lysine demethylases (KDM) are responsible for histone demethylation and are involved in gene expression regulation. Previous studies have shown that histone lysine demethylation plays an important role in gonadal development of vertebrates. The KDM family consists of eight subfamilies, i.e., kdm1, kdm2, kdm3, kdm4, kdm5, kdm6, kdm7 and JmjC-only subfamily. In this study, 13 to 63 KDM genes in 23 representative species were identified based on the available version of genome assembly. Phylogenetic relationships, domain architecture, and synteny of these genes were comprehensively analyzed and the results suggested KDM genes probably originated from the early diverging metazoan and significantly expanded in vertebrates with multiple whole genome duplication, especially in the third-round whole genome duplication (3R-WGD) and polyploidization of teleosts. The subfamilies of kdm2, kdm3, kdm4, kdm5, kdm6 and kdm7 were duplicated with 1R-2R events, and duplicates of kdm2a, kdm4a, kdm5b and kdm6b were resulted from 3R-WGD. Based on transcriptome data, the KDM genes were found to be dominantly expressed in the ovary and testis. More than 80% of KDM genes displayed sexual dimorphic expression, with 15 genes dominantly expressed in ovaries, and 12 genes dominantly expressed in testes. Importantly, from transcriptome data, qRT-PCR and fluorescence in situ hybridization during sex reversal, genes with higher expression in ovary than testis, such as kdm1b and two JmjC-only subfamily members hspbap1 and riox1, were downregulated, while other genes, such as kdm3c, kdm5bb, kdm6ba, kdm6bb and kdm7b, with higher expression in testis than ovary, were upregulated in ovotestis, indicating these genes play critical roles in the gonadal development and sex reversal. This study provided new insights into the evolution of the KDM genes and a fundamental clue for understanding their important roles in sex differentiation and gonadal development in teleosts.

Functional analysis of a SoxE gene in the oriental freshwater prawn, Macrobrachium nipponense by molecular cloning, expression pattern analysis, and in situ hybridization (de Haan, 1849).

In this study, a full-length cDNA sequence of SoxE (subgroup E within the Sox family of transcription factors) was cloned from Macrobrachium nipponense and named MnSoxE1. The full-length cDNA of MnSoxE1 is 1748 bp, consisting of a 110 bp 5' UTR, a 105 bp 3' UTR, and a 1533 bp ORF that encodes 510 amino acids. Conserved domains showed that MnSoxE1 has a high similarity to the SoxE gene of Penaeus vannamei. Phylogenetic tree analysis classified that MnSoxE1 with the SoxE gene of other arthropods into one clade. These results suggested that MnSoxE1 belongs to the SoxE subgroup. During embryonic development, MnSoxE1 was mainly expressed in the gastrula stage, implicating its involvement in tissue cell differentiation and formation. In the post-larval stages, the expression of MnSoxE1 continued to increase on days 1-10. The expression level in males was significantly higher than that in females. Males are clearly distinguishable from females on post-larval day 25, showing that MnSoxE1 may play a role in promoting early development and germ cell and gonadal differentiation, especially for males. qPCR analysis showed that MnSoxE1 may also be involved in oogonium proliferation during ovary development. Further in situ hybridization analysis revealed that MnSoxE1 was mainly located in oocytes and spermatocytes, especially in sertoli cells, and implies that it may be involved in the development of oocytes and spermatocytes, as well as the maintenance of testes in mature prawns. These results indicate that MnSoxE1 is involved in gonadal differentiation and development in M. nipponense, especially testis development.

Genome-wide identification, evolution of ATF/CREB family and their expression in Nile tilapia.

The ATF/CREB family of transcription factors represents a large group of basic region-leucine zipper (bZip) proteins that regulate diverse cellular responses. Here we carried out a comprehensive analysis of ATF/CREB family members in 22 representative animal species. The family probably originated from the early diverging metazoan and significantly expanded in vertebrates due to multiple whole genome duplication. Duplicates of atf6 were derived from 2R, and duplicates of creb1, crem, jdp2, creb5, atf4, atf5 and atf7 were products of 3R. We also isolated 21 ATF/CREBs, belonging to 6 subfamilies from Nile tilapia. Based on transcriptome data, most members were found to be dominantly expressed in the head kidney, heart, brain and testis. Some ATF/CREBs displayed sexual dimorphic expression in gonad at 5, 90 and 180 dah (days after hatching), but not at 30 dah. creb1a and atf4a were found to be expressed mainly in phase I and II oocytes of the ovary; while creb1b and atf4b mainly in spermatogenic cells of the testis, indicating divergence of duplicated genes from 3R which suggested neofunctionalization or subfunctionalization in gonad. This is the first genome-wide screening and evolutionary analysis of ATF/CREB family in different animals, particularly in teleosts. The expression analysis of this family in tilapia gonad provided a fundamental clue for understanding their important roles in sex differentiation and gonadal development in teleosts.

Molecular cloning, expression pattern analysis, and in situ hybridization of a Transformer-2 gene in the oriental freshwater prawn, Macrobrachium nipponense (de Haan, 1849).

In this study, we isolated a full-length cDNA sequence from Macrobrachium nipponense and investigated its gene function. We named the gene Mntra-2a because of high similarities and close evolutionary divergence with arthropod tra-2. The full-length cDNA of Mntra-2a was 1293 bp, consisting of a 212 bp 5' UTR, a 268 bp 3' UTR, and an ORF of 813 bp encoding 270 amino acids. It contained an RNA recognition motif and a linker region. Real-time PCR analysis showed that Mntra-2a was highly expressed in the gonads of both males and females. Further in situ hybridization analysis showed that Mntra-2a was mainly located in oocytes and spermatocytes. During embryogenesis, Mntra-2a expression was higher in the cleavage and nauplius stages. During the ovarian reproductive cycle, Mntra-2a expression reached a peak at OvaryV and decreased to the lowest level at OvaryIV. These results indicated that Mntra-2a probably played important roles in embryonic development and early gonad development in M. nipponense. Our results provide basic information for further functional studies of tra-2 in M. nipponense.

Genome-wide identification, evolution of DNA methyltransferases and their expression during gonadal development in Nile tilapia.

DNA methyltransferases (dnmts) are responsible for DNA methylation and play important roles in organism development. In this study, seven dnmts genes (dnmt1, dnmt2, dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1, dnmt3bb.2) were identified in Nile tilapia. Comprehensive analyses of dnmts were performed using available genome databases from representative animal species. Phylogenetic analysis revealed that the dnmts family were highly conserved in teleosts. Based on transcriptome data from eight adult tilapia tissues, the dnmts were found to be dominantly expressed in the head kidney, testis and ovary. Analyses of the gonadal transcriptome data in different developmental stages revealed that all dnmts were expressed in both ovary and testis, and four de novo dnmts (dnmt3aa, dnmt3ab, dnmt3bb.1, dnmt3bb.2) showed higher expression in the testis than in the ovary. Furthermore, during sex reversal induced by Fadrozole, the expression of these four de novo dnmts increased significantly in treated group compared to female control group. By in situ hybridization, the seven dnmts were found to be expressed mainly in phase I and II oocytes of the ovary and spermatocytes of the testis. When gonads were incubated with a methyltransferase inhibitor (5-AzaCdR) in vitro, the expression of dnmts genes were down-regulated significantly, while the expression of cyp19a1a (a key gene in female pathway) and dmrt1 (a key gene in male pathway) increased significantly. Our results revealed the conservation of dnmts during evolution and indicated a potential role of dnmts in epigenetic regulation of gonadal development.

[Cloning and expression of Bmintegrin ß1 in silkworm Bombyx mori].

Integrins are transmembrane glycoproteins, closely related to many physiological and pathological processes. In order to explore its role in silkworm, by PCR and Rapid-amplification of cDNA ends (RACE) technology, the full-length cDNA of Bmintegrin ß1 in silkworm was acquired. The domain was predicted by domain prediction website. Phylogenetic tree was constructed to analyze its evolutionary relationship. By prokaryotic expression system, protein purification method and immunizing mouse, the antibody against Bmintegrin ß1 recombinant protein was obtained. The spatial-temporal expression profile of Bmintegrin ß1 was investigated by semi quantitative PCR and Western blotting. Then we identified all 3 different spliceosomes, and they shared a common open reading frame of 2 502 bp, encoding 833 amino acids. Bmintegrin ß1 contained all the classic domains of the integrin family, such as Integrin-B-tail, transmembrane domain etc. Phylogenetic analysis indicated that Bmintegrin ß1 was close to the homologous proteins from Heliothis assulta and Danaus plexippus. In order to understand the function of Bmintegrin ß1 further, we generated the antibody. In addition, Western blotting demonstrated that the antibody recognized the Bmintegrin ß1 recombinant protein. Then, semi quantitative PCR and Western blotting results showed that Bmintegrin ß1 was widely expressed in most of tissues, among of them, it's exhibited the highest expression level in hemacyte. Overall, this study provides a foundation for the study of silkworm integrin family.

Genome-wide identification, evolution of chromobox family genes and their expression in Nile tilapia.

Chromobox (Cbx) family proteins are transcriptional repressors that involved in epigenetic and developmental processes. In this study, comprehensive analyses of Cbxs were performed using available genome databases from representative animal species. The Cbx family were originated from one Polycomb (Pc) gene like the yeast Pc, which duplicated into two and gave rise to the Pc and the Heterochromatin protein 1 (Hp1) identified in invertebrates from protozoon to lancelet. Rapid expansion of Cbx family members was observed in vertebrates as ~8 (5 Pc and 3 Hp1) were identified in spotted gar, coelacanth and tetrapods. Further expansion of the members to ~14 (9 Pc and 5 Hp1) was observed in teleosts due to the third round genome duplication (3R). Based on transcriptome data from eight adult tilapia tissues, most of the Cbxs were found to be dominantly expressed in the brain, testis, ovary and heart. Analyses of the gonadal transcriptome data from four developmental stages revealed that all Cbxs were expressed in both ovary and testis except Cbx7b, with significant increase of the total and average RPKM from 5 to 90dah (days after hatching). By in situ hybridization, the three most highly and sexual dimorphically expressed Cbx genes in gonads, Cbx1b, Cbx3a and Cbx5, were found to be expressed in phase I and II oocytes of the ovary, and in secondary spermatocytes (Cbx1b and Cbx3a) and spermatids (Cbx5) of the testis. Our results revealed the evolution of Cbx genes and indicated a potential role of Cbxs in epigenetic regulation of gametogenesis.

Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses.

Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide, and was demonstrated to belong to an important house-keeping protein family that extends across eukaryotes and some prokaryotes. In plants, the Arabidopsis ACBP family consists of six AtACBPs (AtACBP1 to AtACBP6), and has been investigated using gene knock-out mutants and overexpression lines. Herein, recent findings on the AtACBPs are examined to provide an insight on their functions in various plant developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid metabolism, with focus on their interaction with long to very-long-chain (VLC) acyl-CoA esters and their potential role in the formation of lipid droplets in seeds and vegetative tissues are discussed. In addition, recent findings on the rice ACBP family are presented. The similarities and differences between ACBPs from Arabidopsis and rice, that represent eudicot and monocot model plants, respectively, are analyzed and the evolution of plant ACBPs by phylogenetic analysis reviewed. Finally, we propose potential uses of plant ACBPs in phytoremediation and in agriculture related to the improvement of environmental stress tolerance and seed oil production.

Identification and expression profile analysis of NUCLEAR FACTOR-Y families in Physcomitrella patens.

NUCLEAR FACTOR Y transcription factors belong to a multimember family and consist of NF-YA/B/C subunits. Members of the NF-Y family have been reported to regulate physiological processes in plant. In this study, we identified and annotated two NF-YA, nine NF-B, and twelve NF-YC proteins in the genome of Physcomitrella patens. Analyses of conserved domains demonstrated that PpNF-YA/B/C shared the same conserved domains with their orthologous proteins in Arabidopsis, O. sativa and mouse. Expression profiles indicated that PpNF-Ys were widely expressed in different tissues and developmental stages of P. patens throughout protonema and gametophores. The majority of PpNF-Y genes were responsive to abiotic stress via either ABA-independent or -dependent pathways. Some of ABA-regulated PpNF-Y expression were mediated by ABI3. To our knowledge, this study was the first to evaluate NF-Y families in Physcomitrella patens, and provides a foundation to dissect the function of PpNF-Ys.