Transcriptome analysis reveals the synergistic involvement of MGLL and LPIN1 in fatty acid synthesis in broiler pectoral muscles.

Fatty acids (FAs) are one of the most important bioactive compounds affecting the quality of meat. In this study, we compared the expression profiles of genes involved in FA production in the breast muscle of Jingxing Yellow chickens at different days of age determined by transcriptomic analysis to identify key genes and pathways regulating the FA composition of the breast muscle. Through clustering analysis of gene expression data, the growth process of broiler chickens can be divided into two stages, namely the growth and development stage at the 35th and 63rd days of age (D35, D63), and the mature stage at the 119th day of age (D119). The content of some important unsaturated fatty acids (UFAs), such as C18:2n6c, C20:4n6, and C22:6n3, in the pectoral muscles, differed significantly between these two stages (p < 0.05). Therefore, we compared the gene expression profiles at D35 and D63 with those at D119, and identified differentially expressed genes (DEGs). The gene modules related to the five UFAs with significant changes were identified by weighted gene co-expression network analysis (WGCNA), and then 150 crossover genes were identified by crossover analysis of the detected DEGs and WGCNA. The results of the pathway enrichment analysis revealed the glycerolipid metabolism pathway related to lipid metabolism, in which the MGLL and LPIN1 genes were particularly enriched. In this study, the expression levels of MGLL and LPIN1 showed an increasing trend during the growth process of broilers, with a negative regulatory effect on the significantly reduced content of C18:2n6c in the pectoral muscle, and a positive regulatory effect on the significantly increased content of C20:4n6. These findings indicated that MGLL and LPIN1 synergistically promote the deposition of FAs, which may further promote the conversion of linoleic acid (C18:2n6c) to arachidonic acid (C20:4n6). Therefore, screening and identifying FA production-related functional genes are key to elucidate the regulatory molecular mechanism of production of FAs in chicken muscle, aiming to provide a theoretical basis for improving chicken meat quality.

Quantitative Trait Loci Sequencing and Genetic Mapping Reveal Two Main Regulatory Genes for Stem Color in Wax Gourds.

Stem color is an important agronomic trait of wax gourds. However, its regulatory genes have not been identified. In this study, 105 inbred lines constructed from two parents (GX-71 and MY-1) were sequenced and quantitative trait loci sequencing was used to mine the genes that regulate stem color in wax gourds. The results identified two quantitative trait loci related to stem color, qSC5 and qSC12, located on Chr05 (11,134,567-16,459,268) and Chr12 (74,618,168-75,712,335), respectively. The qSC5 had a phenotypic variation rate of 36.9% and a maximum limit of detection of 16.9. And the qSC12 had a phenotypic variation rate of 20.9%, and a maximum limit of detection of 11.2. Bch05G003950 (named BchAPRR2) and Bch12G020400 were identified as candidate genes involved in stem color regulation in wax gourds. The chlorophyll content and expression of BchAPRR2 and Bch12G020400 were significantly higher in green-stemmed wax gourds than in white-stemmed ones. Therefore, BchAPRR2 and Bch12G020400 were considered the main and secondary regulatory genes for wax gourd stem color, respectively. Finally, InDel markers closely linked to BchAPRR2 were developed to validate the prediction of wax gourd stem color traits in 55 germplasm lines, with an accuracy of 81.8%. These findings lay the foundation for exploring the genetic regulation of wax gourd stem color and future research on wax gourd breeding.

Effect of myristic acid supplementation on triglyceride synthesis and related genes in the pectoral muscles of broiler chickens.

Fatty acids (FAs) can serve as energy for poultry, maintain normal cell structure and function, and support a healthy immune system. Although the addition of polyunsaturated fatty acids (PUFAs) to the diet has been extensively studied and reported, the mechanism of action of saturated fatty acids (SFAs) remains to be elucidated. We investigated the effect of 0.04% dietary myristic acid (MA) on slaughter performance, lipid components, tissue FAs, and the transcriptome profile in chickens. The results showed that dietary MA had no effect on slaughter performance (body weight, carcass weight, eviscerated weight, and pectoral muscle weight) (P > 0.05). Dietary MA enrichment increased MA (P < 0.001) and triglycerides (TGs) (P < 0.01) levels in the pectoral muscle. The levels of palmitic acid, linoleic acid (LA), arachidonic acid (AA), SFAs, monounsaturated fatty acids (MUFAs), and PUFAs were significantly higher (P < 0.01) in the MA supplementation group compared to the control group. However, there were no significant differences in the ratios of PUFA/SFA and n6/omega-3 (n3) between the two groups. The MA content was positively correlated with the contents of palmitic acid, LA, linolenic acid (ALA), n3, n6, SFAs, and unsaturated fatty acids (UFA). DHCR24, which is known to be involved in steroid metabolism and cholesterol biosynthesis pathways, was found to be a significantly lower in the MA supplementation group compared to the control group (P < 0.05, log2(fold change) = -0.85). Five overlapping co-expressed genes were identified at the intersection between the differential expressed genes and Weighted Gene Co­expression Network Analysis-derived hub genes associated with MA phenotype, namely BHLHE40, MSL1, PLAGL1, SRSF4, and ENSGALG00000026875. For the TG phenotype, a total of 28 genes were identified, including CHKA, KLF5, TGIF1, etc. Both sets included the gene PLAGL1, which has a negative correlation with the levels of MA and TG. This study provides valuable information to further understand the regulation of gene expression patterns by dietary supplementation with MA and examines at the molecular level the phenotypic changes induced by supplementation with MA.

Screening for brown-spot disease and drought stress response and identification of dual-stress responsive genes in rice cultivars of Northeast India.

Rice cultivation in Northeast India (NEI) primarily relies on rainfed conditions, making it susceptible to severe drought spells that promote the onset of brown spot disease (BSD) caused by Bipolaris oryzae. This study investigates the response of prevalent rice cultivars of NEI to the combined stress of drought and B. oryzae infection. Morphological, physiological, biochemical, and molecular changes were recorded post-stress imposition. Qualitative assessment of reactive oxygen species through DAB (3,3-diaminobenzidine) assay confirmed the elicitation of plant defense responses. Based on drought scoring system and biochemical analyses, the cultivars were categorized into susceptible (Shasharang and Bahadur), moderately susceptible (Gitesh and Ranjit), and moderately tolerant (Kapilee and Mahsuri) groups. Antioxidant enzyme accumulation (catalase, guaiacol peroxidase) and osmolyte (proline) levels increased in all stressed plants, with drought-tolerant cultivars exhibiting higher enzyme activities, indicating stress mitigation efforts. Nevertheless, electrolyte leakage and lipid peroxidation rates increased in all stressed conditions, though variations were observed among stress types. Based on findings from a previous transcriptomic study, a total of nine genes were chosen for quantitative real-time PCR analysis. Among these, OsEBP89 appeared as a potential negative regulatory gene, demonstrating substantial upregulation in the susceptible cultivars at both 48 and 72 h post-treatment (hpt). This finding suggests that OsEBP89 may play a role in conferring drought-induced susceptibility to BSD in the rice cultivars being investigated. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01447-4.

Strategies for the Enhancement of Secondary Metabolite Production via Biosynthesis Gene Cluster Regulation in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae (A. oryzae) has been extensively used for the biosynthesis of numerous secondary metabolites with significant applications in agriculture and food and medical industries, among others. However, the identification and functional prediction of metabolites through genome mining in A. oryzae are hindered by the complex regulatory mechanisms of secondary metabolite biosynthesis and the inactivity of most of the biosynthetic gene clusters involved. The global regulatory factors, pathway-specific regulatory factors, epigenetics, and environmental signals significantly impact the production of secondary metabolites, indicating that appropriate gene-level modulations are expected to promote the biosynthesis of secondary metabolites in A. oryzae. This review mainly focuses on illuminating the molecular regulatory mechanisms for the activation of potentially unexpressed pathways, possibly revealing the effects of transcriptional, epigenetic, and environmental signal regulation. By gaining a comprehensive understanding of the regulatory mechanisms of secondary metabolite biosynthesis, strategies can be developed to enhance the production and utilization of these metabolites, and potential functions can be fully exploited.

Identification and Analysis of lncRNA and circRNA Related to Wheat Grain Development.

The role of lncRNA and circRNA in wheat grain development is still unclear. The objectives of this study were to characterize the lncRNA and circRNA in the wheat grain development and to construct the interaction network among lncRNA, circRNA, and their target miRNA to propose a lncRNA-circRNA-miRNA module related to wheat grain development. Full transcriptome sequencing on two wheat varieties (Annong 0942 and Anke 2005) with significant differences in 1000-grain weight at 10 d (days after pollination), 20 d, and 30 d of grain development were conducted. We detected 650, 736, and 609 differentially expressed lncRNA genes, and 769, 1054, and 1062 differentially expressed circRNA genes in the grains of 10 days, 20 days and 30 days after pollination between Annong 0942 and Anke 2005, respectively. An analysis of the lncRNA-miRNA and circRNA-miRNA targeting networks reveals that circRNAs exhibit a more complex and extensive interaction network in the development of cereal grains and the formation of grain shape. Central to these interactions are tae-miR1177, tae-miR1128, and tae-miR1130b-3p. In contrast, lncRNA genes only form a singular network centered around tae-miR1133 and tae-miR5175-5p when comparing between varieties. Further analysis is conducted on the underlying genes of all target miRNAs, we identified TaNF-YB1 targeted by tae-miR1122a and TaTGW-7B targeted by miR1130a as two pivotal regulatory genes in the development of wheat grains. The quantitative real-time PCR (qRT-PCR) and dual-luciferase reporter assays confirmed the target regulatory relationships between miR1130a-TaTGW-7B and miR1122a-TaNF-YB1. We propose a network of circRNA and miRNA-mediated gene regulation in the development of wheat grains.

Three new LmbU targets outside lmb cluster inhibit lincomycin biosynthesis in Streptomyces lincolnensis.

BACKGROUND: Antibiotics biosynthesis is usually regulated by the cluster-situated regulatory gene(s) (CSRG(s)), which directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). Previously, we have demonstrated that LmbU functions as a cluster-situated regulator (CSR) of lincomycin. And it has been found that LmbU regulates twenty non-lmb genes through comparative transcriptomic analysis. However, the regulatory mode of CSRs' targets outside the BGC remains unknown. RESULTS: We screened the targets of LmbU in the whole genome of Streptomyces lincolnensis and found fourteen candidate targets, among which, eight targets can bind to LmbU by electrophoretic mobility shift assays (EMSA). Reporter assays in vivo revealed that LmbU repressed the transcription of SLINC_0469 and SLINC_1037 while activating the transcription of SLINC_8097. In addition, disruptions of SLINC_0469, SLINC_1037, and SLINC_8097 promoted the production of lincomycin, and qRT-PCR showed that SLINC_0469, SLINC_1037, and SLINC_8097 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. CONCLUSIONS: LmbU can directly regulate genes outside the lmb cluster, and these genes can affect both lincomycin biosynthesis and the transcription of lmb genes. Our results first erected the cascade regulatory circuit of LmbU and regulators outside lmb cluster, which provides the theoretical basis for the functional research of LmbU family proteins.

Production improvement of an antioxidant in cariogenic Streptococcus mutans UA140.

AIMS: This study aimed to improve the production of mutantioxidin, an antioxidant encoded by a biosynthetic gene cluster (mao) in Streptococcus mutans UA140, through a series of optimization methods. METHOD AND RESULTS: Through the construction of mao knockout strain S. mutans UA140∆mao, we identified mutantioxidin as the antioxidant encoded by mao and verified its antioxidant activity through a reactive oxygen species (ROS) tolerance assay. By optimizing the culture medium and fermentation time, 72 h of fermentation in chemically defined medium (CDM) medium was determined as the optimal fermentation conditions. Based on two promoters commonly used in Streptococcus (ldhp and xylS1p), eight promoter refactoring strains were constructed, nevertheless all showed impaired antioxidant production. In-frame deletion and complementation experiments demonstrated the positive regulatory role of mao1 and mao2, on mao. Afterward, the mao1 and mao2, overexpression strain S. mutans UA140/pDL278:: mao1mao2, were constructed, in which the production of mutantioxidin was improved significantly. CONCLUSIONS: In this study, through a combination of varied strategies such as optimization of fermentation conditions and overexpression of regulatory genes, production of mutantioxidin was increased by 10.5 times ultimately.

Three new polyketides from vasR2 gene over-expressed mutant strain of Verrucosispora sp. NS0172 / 中国天然药物

Over-expression of the pathway specific positive regulator gene is an effective way to activate silent gene cluster. In the curret study, the SARP family regulatory gene, vasR2, was over-expressed in strain Verrucosispora sp. NS0172 and the cryptic gene cluster responsible for the biosynthesis of pentaketide ansamycin was partially activated. Two tetraketides (1 and 2) and a triketide (3) ansamycins, together with five known compounds (4-8), were isolated and elucidated from strain NS0172OEvasR2. Their NMR data were completely assigned by analysis of their HR-ESI-MS and

Immunogenicity of DNA vaccines encoding structural proteins and regulatory/accessory proteins derived from an HIV-1 CRF01_AE isolate circulating in China / 中华微生物学和免疫学杂志

Objective To construct two DNA vaccines encoding Gag-Env fusion protein and Tat-Rev-Integrase(C-half)-Vif-Nef fusion protein derived from the first HIV-1 CRF01_AE isolate(AE2f) in Chi-na and to evaluate the immunogenicity in mice. Methods Two DNA vaccines were constructed by inserting the codon optimized and synthesized gag-env fusion gene and tat-rev-integrase(c-half)-vif-nef fusion gene de-rived from AE2f into mammalian expression vector pDRVISV1. 0, the generated DNA vaccines were desig-nated as pSVAE/GE and pSVAE/TRIVN, respectively, and their in vitro expression were determined by Western blot with transfected 293T cells. Mice were i. m. immunized with either pDRVI1.0 as mock control, pSVAE/GE or pSYAE/TRIVN for 4 times at two-week interval. Two weeks following the final im-munization, cellular responses to pool of HIV-1 Env, Gag, Tat, Rev, Intergrase, Vif and Nef peptides were evaluated by ELISPOT assay. Results The construction of DNA vaccine pSVAE/GE and pSVAE/TRIVN was validated by restriction enzyme digestion and bidirectional sequencing. Western blot showed a specific band at molecular mass 220×10~3 in lane of pSVAE/GE transfeeted 293T cell and a specific band at 95×10~3 in the lane of pSVAE/TRIVN. Both DNA vaccines mounted significant specific T cell responses with (3010 ± 566) SFC/10~6 splenocytes for DNA vaccine pSV AE/GE and (948 ± 737) SFC/10~6 spleno-cytes for DNA vaccine pSVAE/TRIVN, whereas the mock control of pDRVISV1.0 only raised marginal T cell responses. Conclusion Both pSVAE/GE and pSVAE/TRIVN were capable of expressing the inserted fusion immunogen genes and able to elicit vigorous cellular immune responses, therefore, these DNA vac-cines are highly immunogenic.

Application of Tn5 Transposon Mutagenesis Technology in Molecular and Genetic Researches of Gram-negative Bacteria / 中国生物工程杂志

With development of wide-host-range vector systems,Tn5 transposon and its derivative vectors have been widely applied to genetic research of gram-negative bacteria.The applications of Tn5 transposon mutagenesis technology to genetic researches of bacteria were briefly discussed,including researches on biological control mechanisms of biocontrol bacteria,identification of bacterial essential genes,discovering virulence genes of bacterial pathogens,characterization of metabolism regulatory genes and genetic improvements of bacteria.

Expression of Several Cell Cycle Regulatory Genes in Cultured AT-1 Cardiomyocytes after Formation of Synchronously Beating Sheets

BACKGROUND AND OBJECTIVE: AT-1 cells have been derived from the left atrial tissue in which the ANF promoter targeted SV40 large T antigen expression. When cultured, clusters of spontaneously contracting cells were observed after 4-5 days and contiguous sheets of synchronously beating cardiomyocytes were formed after 10 days. In this study, expression of several cell cycle regulatory genes were monitored through Northern blot analyses in AT-1 cells during beating and after formation of beating sheets (BS). MATERIALS AND METHOD: AT-1 RNAs were obtained in 3 days after plating, during beating and after formation of BS, and used for Northern blot analyses. RESULTS: alpha-Cardiac myosin heavy chain expression was prominent in beating cells, as would be expected for this contractile protein isoform but ANF was decreased after beating. Gax was not expressed in cultured AT-1 cells but in AT-1 tumor and murine heart. p53 and p21 were decreased after beating which indicate transcription level of p53 and p21 correlated well in AT-1 cells. In contrast, pRB and p107 were increased after beating but p68 (2.4 kb) which arose by alternative splicing of p107 and lacks the pocket domain B was decreased in beating cells. pTCS2, murine tuberous sclerosis gene, represented similar levels during beating but a little was decreased after formation of BS. mRAD50, the murine homologue of yeast DNA recombinational repair gene RAD50, was increased in beating cells, a similar pattern to p107 and pRB. But the p50 arose by alternative splicing of mRAD50 and has 3' half of mRAD50 had unexpectedly appeared and maintained after beating. CONCLUSION: The expression of cell cycle regulatory genes after beating and formation of BS in AT-1 cells showed gene-specific pattern and the p50 which has homology to the mRAD50 may participate in differentiation of cardiomyocytes.