CDS Region Research Articles

Cloning, bioinformatics analysis and expression of the cysteine dioxygenase type 1 (CDO1) gene in domestic yak.

The CDO1 gene is an important gene in the taurine synthesis pathway and has been observed to have high expression in ovaries of female mammals. This study aims to explore the conservation of CDO1 gene in domestic yaks, as well as to examine the fundamental characteristics of CDO1 gene and its expression in female yaks. Ovarian samples were collected from yaks in the follicular phase, luteal phase and gestation period in this experiment, and their total RNA and protein were extracted. Then Polymerase Chain Reaction (PCR) and bioinformatics online software were used to clone and analyze the CDO1 gene. The relative expression of CDO1 in yak ovaries was detected by Quantitative Real-time PCR (RT-qPCR) and Western blotting. The distribution and localization of CDO1 protein in ovary were detected by immunohistochemistry. We have successfully cloned the coding region of CDO1 gene in yak. The results showed that the CDS region of CDO1 gene was 603 bp, encoding 200 amino acids, and was a relatively stable hydrophilic protein. CDO1 is relatively conservative in species evolution. The protein encoded by CDO1 gene does not have a signaling peptide or a transmembrane structure. It is a protein that is not involved in transmembrane transport and is mainly located in the cytoplasm. The secondary structure of the protein is dominated by the random coil. CDO1 is estimated to interact with 10 proteins. The results of RT-qPCR and Western blotting showed that the CDO1 gene exhibited the highest expression in the ovary during the luteal phase and the lowest expression in the ovary during the follicular phase (P < 0.01). The results of immunohistochemistry showed that CDO1 was mainly expressed in granular cells, theca cells and lutein cells of ovarian tissue. These results suggest that the CDO1 gene has undergone minimal evolutionary changes during the course of animal evolution. The results provide a reference for further investigation of the function of CDO1 gene in reproduction and production in yaks.

Molecular Characterizations of FAM13A and Its Functional Role in Inhibiting the Differentiation of Goat Intramuscular Adipocytes through RIG-I Receptor Signaling Pathway.

The aim of this study was to elucidate the effect of FAM13A on the differentiation of goat intramuscular precursor adipocytes and its mechanism of action. Here, we cloned the CDS region 2094 bp of the goat FAM13A gene, encoding a total of 697 amino acid residues. Functionally, overexpression of FAM13A inhibited the differentiation of goat intramuscular adipocytes with a concomitant reduction in lipid droplets, whereas interference with FAM13A expression promoted the differentiation of goat intramuscular adipocytes. To further investigate the mechanism of FAM13A inhibiting adipocyte differentiation, 104 differentially expressed genes were screened by RNA-seq, including 95 up-regulated genes and 9 down-regulated genes. KEGG analysis found that the RIG-I receptor signaling pathway, NOD receptor signaling pathway and toll-like receptor signaling pathway may affect adipogenesis. We selected the RIG-I receptor signaling pathway enriched with more differential genes as a potential adipocyte differentiation signaling pathway for verification. Convincingly, the RIG-I like receptor signaling pathway inhibitor (HY-P1934A) blocked this pathway to save the phenotype observed in intramuscular adipocyte with FAM13A overexpression. Finally, the upstream miRNA of FAM13A was predicted, and the targeted inhibition of miR-21-5p on the expression of FAM13A gene was confirmed. In this study, it was found that FAM13A inhibited the differentiation of goat intramuscular adipocytes through the RIG-I receptor signaling pathway, and the upstream miRNA of FAM13A (miR-21-5p) promoted the differentiation of goat intramuscular adipocytes. This work extends the genetic regulatory network of IMF deposits and provides theoretical support for improving human health and meat quality from the perspective of IMF deposits.

Functional analysis on the role of HvHKT1.4 in barley (Hordeum vulgare L.) salinity tolerance

High-affinity potassium transporters (HKTs) are well known proteins that govern the partitioning of Na+ between roots and shoots. Six HvHKTs were identified in barley and designated as HvHKT1.1, HvHKT1.3, HvHKT1.4, HvHKT1.5, HvHKT2.1 and HvHKT2.2 according to their similarity to previously reported OsHKTs. Among these HvHKTs, HvHKT1.4 was highly up-regulated under salinity stress in both leaves and roots of Golden Promise. Subcellular localization analysis showed that HvHKT1.4 is a plasma-membrane–localized protein. The knockout mutants of HvHKT1.4 showed greater salinity sensitivity and higher Na+ concentration in leaves than wild-type plants. Haplotype analysis of HvHKT1.4 in 344 barley accessions showed 15 single nucleotide substitutions in the CDS region, belonging to five haplotypes. Significant differences in mean salinity damage scores, leaf Na+ contents and Na+/K+ were found between Hap5 and other haplotypes with Hap5 showing better salinity tolerance. The results indicated that HvHKT1.4 can be an effective target in improving salinity tolerance through ion homeostasis.

Connecting the Dots between GmPERK-1 and Enhanced Grain Weight in Glycine max

Large and distinct families of receptor-like kinases (RLKs) play elemental roles in many fundamental processes of plants. The proline-rich extensin-like receptor kinase (PERK) family is one of the most pivotal classes of RLKs. To date, there have been no comprehensive or published studies conducted on the PERK gene family in Glycine max. This research aimed to characterize the role of the PERK gene family in cultivated soybean using a systematic array of bioinformatic and experimental approaches. We identified 16 PERK members in G. max through local BLASTp, using PERK members from Arabidopsis thaliana as a query. Tissue expression of genes, predicted via tissue specific expression analysis from the soybean database “SoyBase”, revealed that these PERK genes exhibit differentiated expression patterns in various plant organs. The gene structure was predicted via Gene Structure Display Server (GSDS). Phylogeny was demonstrated through an evolutionary tree employing the neighbor-joining method. Subcellular localization of proteins was identified via “Softberry” and cis-acting elements were identified through PlantCARE. The KASP (Kompetitive Allele Specific PCR (KASP)) marker was developed for the GmPERK-1-C and GmPERK-1-T allele, targeting position 167 nt in the CDS region. Genotyping results indicated that GmPERK-1 exhibits promising potential for utilization in molecular breeding programs for soybean to increase crop yield. Collectively, our findings indicate that G. max accessions harboring the GmPERK-1-C allele exhibit significantly higher thousand grain weight compared to accessions carrying the GmPERK-1-T allele. This research enhances the understanding of the molecular roles of PERK genes in G. max, providing valuable insights for the utilization of favorable genetic variations in soybean molecular breeding programs.

Genome Assembly and Structural Variation Analysis of Luffa acutangula Provide Insights on Flowering Time and Ridge Development.

Luffa spp. is an important worldwide cultivated vegetable and medicinal plant from the Cucurbitaceae family. In this study, we report a high-quality chromosome-level genome of the high-generation inbred line SG261 of Luffa acutangula. The genomic sequence was determined by PacBio long reads, Hi-C sequencing reads, and 10× Genomics sequencing, with an assembly size of 739.82 Mb, contig N50 of 18.38 Mb, and scaffold N50 of 56.08 Mb. The genome of L. acutangula SG261 was predicted to contain 27,312 protein-coding genes and 72.56% repetitive sequences, of which long terminal repeats (LTRs) were an important form of repetitive sequences, accounting for 67.84% of the genome. Phylogenetic analysis reveals that L. acutangula evolved later than Luffa cylindrica, and Luffa is closely related to Momodica charantia. Comparing the genome of L. acutangula SG261 and L. cylindrica with PacBio data, 67,128 high-quality structural variations (SVs) and 55,978 presence-absence variations (PAVs) were identified in SG261, resulting in 2424 and 1094 genes with variation in the CDS region, respectively, and there are 287 identical genes affected by two different structural variation analyses. In addition, we found that the transcription factor FY (FLOWERING LOCUS Y) families had a large expansion in L. acutangula SG261 (flowering in the morning) compared to L. cylindrica (flowering in the afternoon), which may result in the early flowering time in L. acutangula SG261. This study provides valuable reference for the breeding of and pan-genome research into Luffa species.

Comparative Cytological and Gene Expression Analysis Reveals That a Common Wild Rice Inbred Line Showed Stronger Drought Tolerance Compared with the Cultivar Rice.

Common wild rice (Oryza rufipogon Griff.) is an important germplasm resource containing valuable genes. Our previous analysis reported a stable wild rice inbred line, Huaye3, which derives from the common wild rice of Guangdong Province. However, there was no information about its drought tolerance ability. Here, we assessed the germination characteristics and seedling growth between the Dawennuo and Huaye3 under five concentrations of PEG6000 treatment (0, 5%, 10%, 15%, and 20%). Huaye3 showed a stronger drought tolerance ability, and its seed germination rate still reached more than 52.50% compared with Dawennuo, which was only 25.83% under the 20% PEG6000 treatment. Cytological observations between the Dawennuo and Huaye3 indicated the root tip elongation zone and buds of Huaye3 were less affected by the PEG6000 treatment, resulting in a lower percentage of abnormalities of cortical cells, stele, and shrinkage of epidermal cells. Using the re-sequencing analysis, we detected 13,909 genes that existed in the genetic variation compared with Dawennuo. Of these genes, 39 were annotated as drought stress-related genes and their variance existed in the CDS region. Our study proved the strong drought stress tolerance ability of Huaye3, which provides the theoretical basis for the drought resistance germplasm selection in rice.

Exploring the Molecular Characteristics and Role of PDGFB in Testis and Epididymis Development of Tibetan Sheep.

Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep.

MiR-25–5p in exosomes derived from UVB-induced fibroblasts regulates melanogenesis via TSC2-dominated cellular organelle dysfunction

BackgroundFew reports have confirmed whether exosomes derived from fibroblasts can regulate the process of melanogenesis. We wondered whether exosomes derived from fibroblasts could have a potent regulatory effect on melanogenesis and explored the underlying mechanisms. ObjectiveThis study aimed to find the role of fibroblasts in melanocytes and revealed the related mechanisms. MethodsRT-qPCR, Western blot analysis were conducted to measure the RNA and protein expression level of various related genes. miRNA sequencing, mass spectrum analysis and subsequent bioinformatics analysis were employed to find the underlying targets. Zebrafish were employed to measure the melanin synthesis related process in vivo. Furthermore, electron microscopy, ROS measurement and dual-luciferase reporter assay were adopted to investigate the relationship between these processes. ResultsWe found that exosomes derived from human primary dermal fibroblasts were internalized by human primary melanocytes and MNT1 cells and that the melanin content and the expression of melanin synthesis-related proteins TYR and MITF was inhibited by exosomes derived from UVB-induced human primary dermal fibroblasts. The miRNA expression profile in secreted exosomes changed significantly, with miR-25–5p identified as capable of regulating TSC2 expression via the CDS region. The miR-25–5p-TSC2 axis could affect the melanin content through subsequent cellular organelle dysfunction, such as mitochondrial dysfunction, endoplasmic reticulum stress and dysregulation of lysosomal cysteine proteases. ConclusionWe unveiled a novel regulatory role of fibroblasts in melanocytes, facilitated by the secretion of exosomes. miR-25–5p within exosomes plays a pivotal role in regulating melanogenesis via TSC2-induced cellular organelle dysfunction.

The demethylase ALKBH5 mediates ZKSCAN3 expression through the m6A modification to activate VEGFA transcription and thus participates in MNNG-induced gastric cancer progression

N-Nitroso compounds (NOCs) are recognized as important factors that promote gastric cancer development, but the specific effects and potential mechanisms by which NOC exposure promotes gastric cancer are still poorly understood. In this study, we explored the effects and potential molecular mechanisms of NOCs on the promotion of gastric cancer using methylnitronitrosoguanidine (MNNG), a classical direct carcinogen of NOC. The results of in vivo and in vitro experiments showed that chronic and low-concentration MNNG exposure significantly promoted the malignant progression of tumors, including cell migration, cell invasion, vasculogenic mimicry (VM) formation, cell spheroid formation, stem cell-like marker expression, and gastric cancer growth and metastasis. Mechanistically, we revealed that demethylase ALKBH5 regulated the level of the N6‑methyladenosine (m6A) modification in the 3′UTR and CDS region of the ZKSCAN3 mRNA to promote ZKSCAN3 expression, mediated the binding of ZKSCAN3 to the VEGFA promoter region to regulate VEGFA transcription, and participated in MNNG-induced gastric cancer cell migration, invasion, VM formation, cell spheroid formation, stem cell-like marker expression and ultimately gastric cancer progression. In addition, our study revealed that ALKBH5-ZKSCAN3-VEGFA signaling was significantly activated during MNNG-induced gastric carcinogenesis, and further studies in gastric cancer patients showed that ALKBH5, ZKSCAN3, and VEGFA expression were upregulated in cancers compared with paired gastric mucosal tissues, that ALKBH5, ZKSCAN3, and VEGFA could serve as important biomarkers for determining patient prognosis, and that the molecular combination showed greater prognostic value. These findings provide a theoretical basis for developing gastric cancer interventions for NOCs and for determining gastric cancer progression.

HSPB1 as an RNA-binding protein mediates the pathological process of osteoarthritis

Heat-shock protein beta1 (HSPB1) is a member of the small HSP family, downregulated in osteoarthritis (OA) chondrocytes and demonstrated the capacity to serve as an RNA-binding protein (RBP). This work aimed to explore the profile of HSPB1 bound RNA and reveal the potential regulation mechanism of HSPB1 in OA. In this work, we captured an unbiased HSPB1-RNA interaction map in Hela cells using the iRIP-seq. The results demonstrated that HSPB1 interacted with plentiful of mRNAs and genomic location toward the CDS region. Functional enrichment of HSPB1-related peaks showed the involvement in gene expression, translation initiation, cellular protein metabolic process, and nonsense-mediated decay. HOMER software analysis showed that HSPB1 bound peaks were over-represented in GAGGAG sequences. In addition, ABLIRC and CIMS algorithm indicated that HSPB1 bound to AU-rich motifs and the proportion of AU-rich peaks in 3′ UTR were slightly higher than that in other regions. Moreover, HSPB1-binding targets analysis revealed several gens were associated with OA including EGFR, PLEC, COL5A1, and ROR2. The association of OA-related mRNAs to HSPB1 was additionally confirmed in OA tissues by the quantitative RIP-PCR experiments. Further experiment demonstrated the downregulation of HSPB1 in OA tissues. In conclusion, our current study confirmed HSPB1 as an RNA-binding protein and revealed its potential function in the pathological process of OA, providing a reliable insight to further investigate the molecular regulation mechanism of HSPB1 in OA.

N6-methyladenosine mRNA methylation positively regulated the response of poplar to salt stress.

As the most abundant form of methylation modification in messenger RNA (mRNA), the distribution of N6-methyladenosine (m6A) has been preliminarily revealed in herbaceous plants under salt stress, but its function and mechanism in woody plants were still unknown. Here, we showed that global m6A levels increased during poplar response to salt stress. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that m6A significantly enriched in the coding sequence region and 3'-untranslated regions in poplar, by recognising the conserved motifs, AGACU, GGACA and UGUAG. A large number of differential m6A transcripts have been identified, and some have been proved involving in salt response and plant growth and development. Further combined analysis of MeRIP-seq and RNA-seq revealed that the m6A hypermethylated and enrich in the CDS region preferred to positively regulate expression abundance. Writer inhibitor, 3-deazaneplanocin A treatment increased the sensitivity of poplar to salt stress by reducing mRNA stability to regulate the expression of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Furthermore, we verified that the methyltransferase PagFIP37 plays a positively role in the response of poplar to salt stress, overexpressed lines have stronger salt tolerance, while RNAi lines were more sensitive to salt, which relied on regulating mRNA stability in an m6A manner of salt-responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Collectively, these results revealed the regulatory role of m6A methylation in poplar response to salt stress, and revealed the importance and mechanism of m6A methylation in the response of woody plants to salt stress for the first time.

Transcriptome-wide map of N6-methyladenosine (m6A) profiling in coronary artery disease (CAD) with clopidogrel resistance

BackgroundClopidogrel resistance profoundly increases the risk of major cardiovascular events in coronary artery disease (CAD) patients. Here, we comprehensively analyse global m6A modification alterations in clopidogrel-resistant (CR) and non-CR patients.MethodsAfter RNA isolation, the RNA transcriptome expression (lncRNA, circRNA, and mRNA) was analysed via RNA-seq, and m6A peaks were identified by MeRIP-seq. The altered m6A methylation sites on mRNAs, lncRNAs, and circRNAs were identified, and then, GO and KEGG pathway analyses were performed. Through joint analysis with RNA-seq and MeRIP-seq data, differentially expressed mRNAs harbouring differentially methylated sites were identified. The changes in m6A regulator levels and the abundance of differentially methylated sites were measured by RT-PCR. The identification of m6A-modified RNAs was verified by m6A-IP-qPCR.ResultsThe expression of 2919 hypermethylated and 2519 hypomethylated mRNAs, 192 hypermethylated and 391 hypomethylated lncRNAs, and 375 hypermethylated and 546 hypomethylated circRNAs was shown to be altered in CR patients. The m6A peaks related to CR indicated lower mark density at the CDS region. Functional enrichment analysis revealed that inflammatory pathways and insulin signalling pathways might be involved in the pathological processes underlying CR. The expression of mRNAs (ST5, KDM6B, GLB1L2, and LSM14B), lncRNAs (MSTRG.13776.1 and ENST00000627981.1), and circRNAs (hsa_circ_0070675_CBC1, hsa-circRNA13011-5_CBC1, and hsa-circRNA6406-3_CBC1) was upregulated in CR patients, while the expression of mRNAs (RPS16 and CREG1), lncRNAs (MSTRG.9215.1), and circRNAs (hsa_circ_0082972_CBC1) was downregulated in CR patients. Moreover, m6A regulators (FTO, YTHDF3, and WTAP) were also differentially expressed. An additional combined analysis of gene expression and m6A peaks revealed that the expression of mRNAs (such as ST5, LYPD2, and RPS16 mRNAs) was significantly altered in the CR patients.ConclusionThe expression of m6A regulators, the RNA transcriptome, and the m6A landscape was altered in CR patients. These findings reveal epitranscriptomic regulation in CR patients, which might be novel therapeutic targets in future.

Genome-wide association mapping combined with gene-based haplotype analysis identify a novel gene for shoot length in rice (Oryza sativa L.)

Key messageGenome-wide association mapping revealed a novel QTL for shoot length across multiple environments. Its causal gene, LOC_Os01g68500, was identified firstly through gene-based haplotype analysis, gene expression and knockout transgenic verification.Strong seedling vigor is an important breeding target for rice varieties used in direct seeding. Shoot length (SL) is one of the important traits associated with seedling vigor characterized by rapid growth of seedling, which enhance seedling emergence. Therefore, mining genes for SL and conducting molecular breeding help to develop varieties for direct seeding. However, few QTLs for SL have been fine mapped or cloned so far. In this study, a genome-wide association study of SL was performed in a diverse rice collection consisting of 391 accessions in two years, using phenotypes generated by different cultivation methods according to the production practice, and a total of twenty-four QTLs for SL were identified. Among them, the novel QTL qSL-1f on chromosome 1 could be stably detected across all three cultivation methods in the whole population and indica subpopulation. Through gene-based haplotype analysis of the annotated genes within the putative region of qSL-1f, and validated by gene expression and knockout transgenic experiments, LOC_Os01g68500 (i.e., Os01g0913100 in RAP-DB) was identified as the causal gene for SL, which has a single-base variation (C-to-A transversion) in its CDS region, resulting in the significant difference in SL of rice. LOC_Os01g68500 encodes a DUF538 (Domain of unknown function) containing protein, and the function of DUF538 protein gene on rice seedling growth is firstly reported in this study. These results provide a new clue for exploring the molecular mechanism regulating SL, and promising gene source for the molecular breeding in rice.

Fine mapping and candidate gene analysis of a novel fertility restorer gene for C-type cytoplasmic male sterility in maize (Zea mays L.).

A novel strong fertility restorer gene Rf12 for C-type cytoplasmic male sterility of maize was finely mapped on chromosome 2. Its best candidate gene Zm00001d007531 is predicted to encode a p-type PPR protein. The lack of strong restorer gene of maize CMS-C greatly limits its application in hybrid seed production. Therefore, the cloning of maize CMS-C novel strong restorer genes is necessary. In this study, a strong restorer line ZH91 for maize CMS-C was found, and the novel restorer gene named Rf12 in ZH91 had been mapped in a 146kb physical interval on maize chromosome 2. Using the third-generation high-throughput sequencing (ONT), the whole genome sequence of ZH91 was got, and with integrating the annotation information of the reference genome B73_RefGen_v4 and B73_RefGen_v5, four candidate genes were predicted in ZH91 within the mapping region. Then using gene cloning, stranded specific RNA sequencing, qRT-PCR analysis and subcellular localization, Zm00001d007531 was identified as the most likely candidate gene of Rf12. Zm00001d007531 encodes a p-type PPR protein with 19 PPR motifs and targets mitochondria and chloroplast. Stranded specific RNA sequencing and qRT-PCR results both show that the expression of Zm00001d007531 between anthers of near-isogenic lines C478Rf12Rf12 and C478rf12rf12 was significantly difference in pollen mother cell stage. And the result of sequence alignment for Zm00001d007531 gene in 60 materials showed that there are twelve SNPs in CDS region of Zm00001d007531 were tightly linked to the fertility. The finding of a novel strong restorer germplasm resource ZH91 for maize CMS-C can greatly promote the application of maize CMS-C line in maize hybrid seeds production, and the identification of candidate gene Zm00001d007531 can accelerate the backcrossing process of maize CMS-C strong restorer gene Rf12 to some extent.

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Improving digestibility of sorghum proteins by CRISPR/Cas9‐based genome editing

AbstractKafirins are the primary storage proteins in sorghum kernels that provide amino acids for seed germination. The highly proteolytic resistant γ‐ and β‐kafirins form the cross‐linked outer layers that encapsulate α‐kafirins to generate protein bodies, resulting in poor digestibility of sorghum grains. The sorghum kafirins thus contribute to the poor quality of the kernels. The nutritional quality and digestibility of sorghum grains can be improved by reducing the contents of kafirin. γ‐Kafirin is encoded by the K2G gene, which is located on sorghum chromosome 2. In this study, we used CRISPR/Cas9 gene editing to target the K2G gene to create new sorghum lines with reduced levels of kafirin. A guide RNA (sgRNA) was designed to introduce mutations in the CDS region that encodes the endoplasmic reticulum signal peptide of γ‐kafirin. The pK2GsgRNA/Cas9 vector was transformed into sorghum using the pollen‐mediated transformation method. Sequencing of the transformants showed that three out of 24 transgenic plants contain genetic mutations in the targeted region. Compared with the wildtype, the γ‐kafirin contents of the mutant plants decreased by 12.75%–19.22%, and the protein digestibility of the mutant kernels increased by 26.91%–74.31% in raw flour. Although the grain weights remained comparable to those of the wildtype, the growth of the mutant plants was more vigorous as the mutant shoots grew taller and thicker compared with those of the wildtype. Our work advances the ability to improve the digestibility of an important crop. The resulting quality improvements can also be rapidly deployed for breeding and generation of transgene‐free, improved cultivars of sorghum, a major crop worldwide.

M6A Reader YTHDC1 Inhibits Ferroptosis and Radiosensitivity by Promoting SREBF1 mRNA Nuclear Export in Nasopharyngeal Carcinoma

Radioresistance is the main reason for nasopharyngeal carcinoma (NPC) recurrence leading to treatment failure, and inducing ferroptosis has gradually been a new way to enhance radiosensitivity. N6-methyladenosine (m6A) is involved in regulation of numerous biological processes. However, whether m6A affects ferroptosis in NPC is still unclear. In this study, we conducted a siRNA library screening to identify m6A reader YTHDC1 as an essential oncogene that suppressed ferroptosis and radiosensitivity by promoting SREBF1 mRNA nuclear export in nasopharyngeal carcinoma. The expression and function of YTHDC1 were assessed via CCK8 cell viability assay, immunostaining, real-time PCR, western blot, radiation clonogenic assay and fluorescence in situ hybridization assay. Ferroptosis was determined by detecting cell viability, lipid peroxidation, abnormal mitochondrial and cell death rate. The in vivo effects of YTHDC1 were examined with RSL3 treatment or lentivirus modification of YTHDC1 expression in radiated mouse models. Based on RSL3-induced ferroptotic cell death model and a siRNA library about m6A modification associated gene screening, we identified m6A reader YTHDC1 could inhibit ferroptosis as well as radiosensitivity of NPC, both in vivo and in vitro. Mechanistically, YTHDC1 protein could recognize m6A sites in the CDS region and 3' untranslated region (3'UTR) of SREBF1 mRNA and promote SREBF1 mRNA nuclear export, which finally resulted in transcriptional upregulation of genes key to ferroptosis such as SCD and FASN. Furthermore, the high expression of YTHDC1 was negatively regulated by ZNF598 via ubiquitination and associated with unfavorable survival in NPC patients due to radioresistance. Our findings reveal the critical role of YTHDC1 specifically in inhibiting ferroptosis and radiosensitivity via m6A-dependent mechanism and provide an exploitable target and therapeutic strategy for overcoming radioresistance in NPC.

Genome-wide analysis of the heat shock transcription factor family reveals saline-alkali stress responses in Xanthoceras sorbifolium.

The heat shock transcription factor (HSF) family is involved in regulating growth, development, and abiotic stress. The characteristics and biological functions of HSF family member in X. sorbifolium, an important oil and ornamental plant, have never been reported. In this study, 21 XsHSF genes were identified from the genome of X. sorbifolium and named XsHSF1-XsHSF21 based on their chromosomal positions. Those genes were divided into three groups, A, B, and C, containing 12, one, and eight genes, respectively. Among them, 20 XsHSF genes are located on 11 chromosomes. Protein structure analysis suggested that XsHSF proteins were conserved, displaying typical DNA binding domains (DBD) and oligomerization domains (OD). Moreover, HSF proteins within the same group contain specific motifs, such as motif 5 in the HSFC group. All XsHSF genes have one intron in the CDS region, except XsHSF1 which has two introns. Promoter analysis revealed that in addition to defense and stress responsiveness elements, some promoters also contained a MYB binding site and elements involved in multiple hormones responsiveness and anaerobic induction. Duplication analysis revealed that XsHSF1 and XsHSF4 genes were segmentally duplicated while XsHSF2, XsHSF9, and XsHSF13 genes might have arisen from transposition. Expression pattern analysis of leaves and roots following salt-alkali treatment using qRT-PCR indicated that five XsHSF genes were upregulated and one XsHSF gene was downregulated in leaves upon NaCl treatment suggesting these genes may play important roles in salt response. Additionally, the expression levels of most XsHSFs were decreased in leaves and roots following alkali-induced stress, indicating that those XsHSFs may function as negative regulators in alkali tolerance. MicroRNA target site prediction indicated that 16 of the XsHSF genes may be regulated by multiple microRNAs, for example XsHSF2 might be regulated by miR156, miR394, miR395, miR408, miR7129, and miR854. And miR164 may effect the mRNA levels of XsHSF3 and XsHSF17, XsHSF9 gene may be regulated by miR172. The expression trends of miR172 and miR164 in leaves and roots on salt treatments were opposite to the expression trend of XsHSF9 and XsHSF3 genes, respectively. Promoter analysis showed that XsHSFs might be involved in light and hormone responses, plant development, as well as abiotic stress responses. Our results thus provide an overview of the HSF family in X. sorbifolium and lay a foundation for future functional studies to reveal its roles in saline-alkali response.

Identification of Loci for Four Important Agronomic Traits in Loose-Curd Cauliflower Based on Genome-Wide Association Studies

Cauliflower is a nutritious vegetable with inflorescences that are specialized to form the edible organs called curds. Uncovering key genes underlying important traits is crucial for the genetic improvement of this important crop. However, the genetic basis of many important agronomic traits, including curd performance and plant architecture in cauliflower, remains unclear. GWASs have proved to be powerful tools to study agronomic traits in many crops. To reveal the genetic basis of four important agronomic traits, namely, the main stem height (MSH), purplish curd (PC), external leaf wing (ELW) and weight of a single curd (WSC), we selected 220 core accessions of loose-curd cauliflower for resequencing, phenotypic investigation and GWAS. The approach revealed significant novel loci. We detected several significant associations: on C02 for MSH and PC, on C06 for ELW and on C01 for WSC. More interestingly, we identified a significant single-peak signal for the weight of a single curd (WSC), an important yield trait, and within this signal interval, we identified the BOB01G136670 gene with five SNPs encoding nonsynonymous mutations in the CDS region; these mutations resulted in two haplotypes with significant differences in curd weight. The weight of a single curd was significantly increased in the varieties with the BOB01G136670 Hap1 allele compared to those with BOB01G136670 Hap2. BOB01G136670 was highly conserved with the homologous genes that encode serine carboxypeptidase and belong to the S10 family in other species, including GS5, which functions as a positive regulator of grain size in rice, wheat and maize. Additionally, BOB01G136670 was highly expressed specifically at the curd enlargement stage, with low or even no expression at all in other tissues and stages, indicating that BOB01G136670 is a plausible candidate gene for WSC. Overall, this study identified genomic loci for four important agronomic traits that are relevant for accelerating biological breeding and the improvement of cauliflower varieties.

Estrogen Receptor Gene 1 (ESR1) Mediates Lipid Metabolism in Goose Hierarchical Granulosa Cells Rather than in Pre-Hierarchical Granulosa Cells.

(1) Background: The role of estrogen receptor gene 1 (ESR1) in female reproduction and lipid metabolism has been extensively investigated. However, its contribution to lipid metabolism during the development of poultry follicles remains unclear. (2) Methods: This study aimed to explore the function of ESR1 via overexpressing (ESR1ov) and interfering (ESR1si) with its expression in pre-hierarchical granulosa cells (phGCs) and hierarchical granulosa cells (poGCs). (3) Results: We successfully cloned and obtained an 1866 bp segment of the full-length CDS region of the Sichuan white goose ESR1 gene. In phGCs of the ESR1ov and ESR1si groups, there were no significant changes compared to the control group. However, in poGCs, the ESR1ov group exhibited decreased lipid deposition, triglycerides, and cholesterol compared to the control group, while the ESR1si group showed increased lipid deposition, triglycerides, and cholesterol. The expression of APOB and PPARα was significantly reduced in the ESR1ov group compared to the ESR1ov-NC group. Moreover, significant changes in the expression of ACCα, DGAT1, SCD, CPT1, and ATGL were observed between the ESR1si and ESR1si-NC group. (4) Conclusions: These findings shed light on the function and molecular mechanism of ESR1 in lipid metabolism in goose poGCs, providing a better understanding of the physiological process of goose follicular development.