Fibronectin 1 supports oocyte in vitro maturation in pigs.

Oocyte in vitro maturation (IVM) based on the follicular fluid (FF) environment can exploit untapped resources, however, what FF factors regulate oocyte maturation remains unclear. This work demonstrated that serum and FF significantly promoted oocyte polar body extrusion (PBE) and subsequent embryo development, and FF was especially effective. Fibronectin 1 (FN1) was predicted as one potential candidate to regulate oocyte maturation by proteomics. FN1 transcription obviously decreased, and the protein expression significantly increased and migrated to plasma membrane or even outside during oocyte IVM. Treatment with 10 ng/mL FN1 significantly improved oocyte PBE rate. FN1 significantly upregulated the percentage of regular spindle morphology, downregulated the γ-H2AX level, decreased the levels of ROS and apoptosis, and increased GSH and mitochondrion contents by ameliorating the expression of corresponding genes. Moreover, FN1 significantly increased the p-PI3K level to enhance the activation of PI3K signaling pathway. In conclusion, this study discovers and confirms that FN1 is one factor in FF that significantly enhances oocyte maturation, and the underlying mechanism is that FN1 ameliorates oocyte nuclear and cytoplasmic maturation by promoting the activation of PI3K signaling pathway.

Rapidly mining candidate cotton drought resistance genes based on key indicators of drought resistance.

BACKGROUND: Focusing on key indicators of drought resistance is highly important for quickly mining candidate genes related to drought resistance in cotton. RESULTS: In the present study, drought resistance was identified in drought resistance-related RIL populations during the flowering and boll stages, and multiple traits were evaluated; these traits included three key indicators: plant height (PH), single boll weight (SBW) and transpiration rate (Tr). Based on these three key indicators, three groups of extreme mixing pools were constructed for BSA-seq. Based on the mapping interval of each trait, a total of 6.27 Mb QTL intervals were selected on chromosomes A13 (3.2 Mb), A10 (2.45 Mb) and A07 (0.62 Mb) as the focus of this study. Based on the annotation information and qRT‒PCR analysis, three key genes that may be involved in the drought stress response of cotton were screened: GhF6'H1, Gh3AT1 and GhPER55. qRT‒PCR analysis of parental and extreme germplasm materials revealed that the expression of these genes changed significantly under drought stress. Cotton VIGS experiments verified the important impact of key genes on cotton drought resistance. CONCLUSIONS: This study focused on the key indicators of drought resistance, laying the foundation for the rapid mining of drought-resistant candidate genes in cotton and providing genetic resources for directed molecular breeding of drought resistance in cotton.

Glyceraldehyde-3-phosphate dehydrogenase Gh_GAPDH9 is associated with drought resistance in Gossypium hirsutum.

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the central enzyme of glycolysis and plays important regulatory roles in plant growth and development and responses to adverse stress conditions. However, studies on the characteristics and functions of cotton GAPDH family genes are still lacking. Methods: In this study, genome-wide identification of the cotton GAPDH gene family was performed, and the phylogeny, gene structures, promoter progenitors and expression profiles of upland cotton GAPDH gene family members were explored by bioinformatics analysis to highlight potential functions. The functions of GhGAPDH9 in response to drought stress were initially validated based on RNA-seq, qRT‒PCR, VIGS techniques and overexpression laying a foundation for further studies on the functions of GAPDH genes. Results: This study is the first systematic analysis of the cotton GAPDH gene family, which contains a total of 84 GAPDH genes, among which upland cotton contains 27 members. Quantitative, phylogenetic and covariance analyses of the genes revealed that the GAPDH gene family has been conserved during the evolution of cotton. Promoter analysis revealed that most cis-acting elements were related to MeJA and ABA. Based on the identified promoter cis-acting elements and RNA-seq data, it was hypothesized that Gh_GAPDH9, Gh_GAPDH11, Gh_GAPDH19 and Gh_GAPDH21 are involved in the response of cotton to abiotic stress. The expression levels of the Gh_GAPDH9 gene in two drought-resistant and two drought-sensitive materials were analyzed by qRT‒PCR and found to be high early in the treatment period in the drought-resistant material. The silencing of Gh_GAPDH9 based on virus-induced gene silencing (VIGS) technology resulted in significant leaf wilting or whole-plant dieback in silenced plants after drought stress compared to the control. The content of-malondialdehyde (MDA) in cotton leaves was significantly increased, and the content of proline (Pro) and chlorophyll (Chl) was reduced. In addition, the leaf wilting and dryness of transgenic lines under drought stress were lower than those of wild-type Arabidopsis, indicating that Gh_GAPDH9 is a positive regulator of drought resistance. In conclusion, our results demonstrate that GAPDH genes play an important role in the response of cotton to abiotic stresses and provide preliminary validation of the function of the Gh_GAPDH9 gene under drought stress. These findings provide an important theoretical basis for further studies on the function of the Gh_GAPDH9 gene and the molecular mechanism of the drought response in cotton.

Using association mapping and local interval haplotype association analysis to improve the cotton drought stress response.

Drought stress has a serious impact on the growth and development of cotton. To explore the relevant molecular mechanism of the drought stress response in cotton, gene mapping based on the QTL interval mapped by simplified genome BSA-seq of the drought-resistance-related RIL population was performed. A QTL region spanning 2.02 Mb on chromosome D07 was selected, and 201 resource materials were genotyped using 9 KASP markers in the interval. After local interval haplotype association analysis, the overlap of the 110 kb peak region confirmed the reliability of this region, and at the same time, the role of GhGF14-30, the only gene in the overlapping region, was modeled in the response of cotton to drought stress. qRTPCR analysis of the materials and population parents proved that this gene plays a role in the drought stress response in cotton. Virus-induced gene silencing proved the importance of this gene in drought-sensitive materials, and drought-resistance-related marker genes also proved that the GhGF14-30 gene may play an important role in the ABA and SOS signaling pathways. This study provides a basis for mining drought stress response functional genes in cotton and lays the foundation for the molecular mechanism of the GhGF14-30 gene in response to drought stress in cotton.

Astaxanthin enhances the development of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes.

Low cloning efficiency limits the wide application of somatic cell nuclear transfer technology. Apoptosis and incomplete DNA methylation reprogramming of pluripotency genes are considered as the main causes for low cloning efficiency. Astaxanthin (AST), a powerfully antioxidative and antiapoptotic carotenoid, is recently shown to improve the development of early embryos, however, the potential role of AST during the development of cloned embryos remains unclear. This study displayed that treating cloned embryos with AST significantly increased the blastocyst rate and total blastocyst cell number in a concentration dependent manner, and also alleviated the damage of H2O2 to the development of cloned embryos. In addition, compared with the control group, AST significantly reduced the apoptotic cell number and rate in cloned blastocysts, and the significantly upregulated expression of anti-apoptotic gene Bcl2l1 and antioxidative genes (Sod1 and Gpx4) and downregulated transcription of pro-apoptotic genes (Bax, P53 and Caspase3) were observed in the AST group. Moreover, AST treatment facilitated DNA demethylation of pluripotency genes (Pou5f1, Nanog and Sox2), in accompany with the improved transcription levels of DNA methylation reprogramming genes (Tet1, Tet3, Dnmt1, Dnmt3a and Dnmt3b) in cloned embryos, and then, the significantly upregulated expression levels of embryo development related genes including Pou5f1, Nanog, Sox2 and Cdx2 were observed in comparison with the control group. In conclusion, these results revealed that astaxanthin enhanced the developmental potential of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes, and provided a promising approach to improve cloning efficiency.

Weighted Gene Co-Expression Network Analysis Reveals Hub Genes for Fuzz Development in Gossypium hirsutum.

Fuzzless Gossypium hirsutum mutants are ideal materials for investigating cotton fiber initiation and development. In this study, we used the fuzzless G. hirsutum mutant Xinluzao 50 FLM as the research material and combined it with other fuzzless materials for verification by RNA sequencing to explore the gene expression patterns and differences between genes in upland cotton during the fuzz period. A gene ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic process, microtubule binding, and other pathways. A weighted gene co-expression network analysis (WGCNA) showed that two modules of Xinluzao 50 and Xinluzao 50 FLM and four modules of CSS386 and Sicala V-2 were highly correlated with fuzz. We selected the hub gene with the highest KME value among the six modules and constructed an interaction network. In addition, we selected some genes with high KME values from the six modules that were highly associated with fuzz in the four materials and found 19 common differential genes produced by the four materials. These 19 genes are likely involved in the formation of fuzz in upland cotton. Several hub genes belong to the arabinogalactan protein and GDSL lipase, which play important roles in fiber development. According to the differences in expression level, 4 genes were selected from the 19 genes and tested for their expression level in some fuzzless materials. The modules, hub genes, and common genes identified in this study can provide new insights into the formation of fiber and fuzz, and provide a reference for molecular design breeding for the genetic improvement of cotton fiber.

Quantitative trait locus mapping and identification of candidate genes for resistance to Verticillium wilt in four recombinant inbred line populations of Gossypium hirsutum.

Improving resistance to Verticillium wilt is of great significance for achieving high and stable yields of Upland cotton (Gossypium hirsutum). To deeply understand the genetic basis of cotton resistance to Verticillium wilt, Verticillium wilt-resistant Upland Lumianyan 28 and four Verticillium wilt-susceptible Acala cotton cultivars were used to create four recombinant inbred line (RIL) populations of 469 families through nested hybridization. Phenotypic data collected in five stressful environments were used to select resistant and sensitive lines and create a mixed pool of extreme phenotypes for BSA-seq. A total of 8 QTLs associated with Verticillium wilt resistance were identified on 4 chromosomes, of which qVW-A12-5 was detected simultaneously in the RIL populations and in one of the RIL populations and was identified for the first time. According to the sequence comparison and transcriptome analysis of candidate genes in the QTL interval between parents and pools, 4 genes were identified in the qVW-A12-5 interval. qRT-PCR of parental and phenotypically extreme lines revealed that Gh_CPR30 was induced by and may be a candidate gene for resistance to Verticillium wilt in G. hirsutum. Furthermore, VIGS technology revealed that the disease severity index (DSI) of the Gh_CPR30-silenced plants was significantly higher than that of the control. These results indicate that the Gh_CPR30 gene plays an important role in the resistance of G. hirsutum to Verticillium wilt, and the study provides a molecular basis for analyzing the molecular mechanism underlying G. hirsutum resistance to Verticillium wilt.

Genome-wide analysis of serine carboxypeptidase-like protein (SCPL) family and functional validation of Gh_SCPL42 unchromosome conferring cotton Verticillium der Verticillium wilt stress in Gossypium hirsutum.

BACKGROUND: Serine carboxypeptidase-like protein (SCPL) plays an important role in response to stress in plant. However, our knowledge of the function of the SCPL gene family is limited. RESULTS: In this study, a comprehensive and systematic analysis of SCPL gene family was conducted to explore the phylogeny and evolution of the SCPL gene in Gossypium hirsutum. The phenotype and molecular mechanism of silencing of the Gh_SCPL42 under Verticillium wilt stress was also studied. Our results showed that 96 SCPL genes were observed in genome of G. hirsutum, which distributed on 25 chromosomes and most of them were located in the nucleus. The phylogenetic tree analysis showed that members of SCPL gene family can be divided into three subgroups in G. hirsutum, which are relatively conservative in evolution. SCPL gene has a wide range of tissue expression types in G. hirsutum. Promoter analysis showed that the most cis-acting elements related to MeJA and ABA were contained. Through RNA-seq combined with genotyping, it was found that 11 GhSCPL genes not only had significant expression changes during Verticillium wilt stress but also had differential SNPs in the upstream, downstream, exonic or intronic regions. The expression of these 11 genes in the resistant (Zhongzhimian 2) and susceptible (Junmian 1) materials was further analyzed by qRT-PCR, it was found that 6 genes showed significant expression differences in the two materials. Among them, Gh_SCPL42 has the most obvious expression change. Furthermore, virus-induced gene silencing (VIGS) showed necrosis and yellowing of leaves and significantly higher disease severity index (DSI) and disease severity rate (DSR) values in VIGS plants than in control silenced Gh_SCPL42 plants. Moreover, the expression levels of genes related to the SA and JA pathways were significantly downregulated. These results show that Gh_SCPL42 might improve resistance to Verticillium wilt through the SA and JA pathways in G. hirsutum. CONCLUSION: In conclusion, our findings indicated that Gh_SCPL42 gene plays an important role in resistance to Verticillium wilt in cotton. It was provided an important theoretical basis for further research on the function of SCPL gene family and the molecular mechanism of resistance to Verticillium wilt in cotton.

Gb_ANR-47 Enhances the Resistance of Gossypium barbadense to Fusarium oxysporum f. sp. vasinfectum (FOV) by Regulating the Content of Proanthocyanidins.

Anthocyanidin reductase (ANR) is an important regulator of flavonoid metabolism, and proanthocyanidins, the secondary metabolites of flavonoids, play an important role in the response of plants to pathogenic stress. Therefore, in this study, the expression analysis of the ANR gene family of Gossypium barbadense after inoculation with Fusarium oxysporum f. sp. vasinfectum (FOV) was performed at different time points. It was found that Gb_ANR-47 showed significant differences in the disease-resistant cultivar 06-146 and the susceptible cultivar Xinhai 14, as well as in the highest root expression. It was found that the expression of Gb_ANR-47 in the resistant cultivar was significantly higher than that in the susceptible cultivar by MeJA and SA, and different amounts of methyl jasmonate (MeJA) and salicylic acid (SA) response elements were found in the promoter region of Gb_ANR-47. After silencing GbANR-47 in 06-146 material by VIGS technology, its resistance to FOV decreased significantly. The disease severity index (DSI) was significantly increased, and the anthocyanin content was significantly decreased in silenced plants, compared to controls. Our findings suggest that GbANR-47 is a positive regulator of FOV resistance in Gossypium barbadense. The research results provide an important theoretical basis for in-depth analysis of the molecular mechanism of GbANR-47 and improving the anti-FOV of Gossypium barbadense.

Genome-wide identification of the DUF668 gene family in cotton and expression profiling analysis of GhDUF668 in Gossypium hirsutum under adverse stress.

BACKGROUND: Domain of unknown function 668 (DUF668) may play a crucial role in the plant growth and developmental response to adverse stress. However, our knowledge of the function of the DUF668 gene family is limited. RESULTS: Our study was conducted based on the DUF668 gene family identified from cotton genome sequencing. Phylogenetic analysis showed that the DUF668 family genes can be classified into four subgroups in cotton. We identified 32 DUF668 genes, which are distributed on 17 chromosomes and most of them located in the nucleus of Gossypium hirsutum. Gene structure and motif analyses revealed that the members of the DUF668 gene family can be clustered in G. hirsutum into two broad groups, which are relatively evolutionarily conserved. Transcriptome data analysis showed that the GhDUF668 genes are differentially expressed in different tissues under various stresses (cold, heat, drought, salt, and Verticillium dahliae), and expression is generally increased in roots and stems. Promoter and expression analyses indicated that Gh_DUF668-05, Gh_DUF668-08, Gh_DUF668-11, Gh_DUF668-23 and Gh_DUF668-28 in G. hirsutum might have evolved resistance to adverse stress. Additionally, qRT-PCR revealed that these 5 genes in four cotton lines, KK1543 (drought resistant), Xinluzao 26 (drought sensitive), Zhongzhimian 2 (disease resistant) and Simian 3 (susceptible), under drought and Verticillium wilt stress were all significantly induced. Roots had the highest expression of these 5 genes before and after the treatment. Among them, the expression levels of Gh_DUF668-08 and Gh_DUF668-23 increased sharply at 6 h and reached a maximum at 12 h under biotic and abiotic stress, which showed that they might be involved in the process of adverse stress resistance in cotton. CONCLUSION: The significant changes in GhDUF668 expression in the roots after adverse stress indicate that GhDUF668 is likely to increase plant resistance to stress. This study provides an important theoretical basis for further research on the function of the DUF668 gene family and the molecular mechanism of adverse stress resistance in cotton.