Saccharomyces cerevisiae is the preferred source of RNA derivatives, which are widely used as supplements for foods and pharmaceuticals. As the most abundant RNAs, the ribosomal RNAs (rRNAs) transcribed by RNA polymerase I (Pol I) have no 5′ caps, thus cannot be translated to proteins. To screen high-nucleic-acid content yeasts more efficiently, a cap-independent protein expression system mediated by Pol I has been designed and established to monitor the regulatory changes of rRNA synthesis by observing the variation in the reporter genes expression. The elements including Pol I-recognized rDNA promoter, the internal ribosome entry site from cricket paralytic virus which can recruit ribosomes internally, reporter genes (URA3 and yEGFP3), oligo-dT and an rDNA terminator were ligated to a yeast episomal plasmid. This system based on the URA3 gene worked well by observing the growth phenotype and did not require the disruption of cap-dependent initiation factors. The fluorescence intensity of strains expressing the yEGFP3 gene increased and drifted after mutagenesis. Combined with flow cytometry, cells with higher GFP level were sorted out. A strain showed 58% improvement in RNA content and exhibited no sequence alteration in the whole expression cassette introduced. This study provides a novel strategy for breeding high-nucleic-acid content yeasts. 相似文献
Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection. 相似文献
Red‐spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis disease, has caused high mortality and heavy economic losses in marine aquaculture worldwide. However, changes in host cell metabolism during RGNNV infection remain largely unknown. Here, the global metabolic profiling during RGNNV infection and the roles of cellular fatty acid synthesis in RGNNV infection were investigated. As the infection progressed, 71 intracellular metabolites were significantly altered in RGNNV‐infected cells compared with mock‐infected cells. The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased, whereas those that correlated with fatty acid synthesis were significantly up‐regulated during RGNNV infection. Among them, tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection. In addition, RGNNV infection induced the formation of lipid droplets and re‐localization of fatty acid synthase (FASN), indicating that RGNNV induced and required lipogenesis for viral infection. The exogenous addition of palmitic acid (PA) enhanced RGNNV infection, and the inhibition of FASN and acetyl‐CoA carboxylase (ACC) significantly decreased RGNNV replication. Additionally, not only inhibition of palmitoylation and phospholipid synthesis, but also destruction of fatty acid β‐oxidation significantly decreased viral replication. These data suggest that cellular fatty acid synthesis and mitochondrial β‐oxidation are essential for RGNNV to complete the viral life cycle. Thus, it has been demonstrated for the first time that RGNNV infection in vitro overtook host cell metabolism and, in that process, cellular fatty acid synthesis was an essential component for RGNNV replication. 相似文献
Kernel weight and morphology are important traits affecting cereal yields and quality. Dissecting the genetic basis of thousand kernel weight (TKW) and its related traits is an effective method to improve wheat yield. In this study, we performed quantitative trait loci (QTL) analysis using recombinant inbred lines derived from the cross ‘PuBing3228 × Gao8901’ (PG-RIL) to dissect the genetic basis of kernel traits. A total of 17 stable QTLs related to kernel traits were identified, notably, two stable QTLs QTkw.cas-1A.2 and QTkw.cas-4A explained the largest portion of the phenotypic variance for TKW and kernel length (KL), and the other two stable QTLs QTkw.cas-6A.1 and QTkw.cas-7D.2 contributed more effects on kernel width (KW). Conditional QTL analysis revealed that the stable QTLs for TKW were mainly affected by KW. The QTLs QTkw.cas-7D.2 and QKw.cas-7D.1 associated with TKW and KW were delimited to the physical interval of approximately 3.82 Mb harboring 47 candidate genes. Among them, the candidate gene TaFT-D1 had a 1 bp insertions/deletion (InDel) within the third exon, which might be the reason for diversity in TKW and KW between the two parents. A Kompetitive Allele-Specific PCR (KASP) marker of TaFT-D1 allele was developed and verified by PG-RIL and a natural population consisted of 141 cultivar/lines. It was found that the favorable TaFT-D1 (G)-allele has been positively selected during Chinese wheat breeding. Thus, these results can be used for further positional cloning and marker-assisted selection in wheat breeding programs. Seventeen stable QTLs related to kernel traits were identified. The stable QTLs for thousand kernel weight were mainly affected by kernel width. TaFT-D1 could be the candidate gene for QTLs QTkw.cas-7D.2 and QKw.cas-7D.1. 相似文献
Atherosclerosis is one of the most common and crucial heart diseases involving the heart and brain. At present, atherosclerosis and its major complications comprise the leading causes of death worldwide. Our purpose was to identify the role of ciRS‐7 in atherosclerosis. Tubulogenesis of HMEC‐1 cell was evaluated utilizing tube formation assay. Cell Counting Kit‐8 assay and flow cytometry were utilized to test viability and apoptosis. Migration assay was utilized to determine the migration capacity of experimental cells. Western blot was applied to examine apoptosis and tube formation‐associated protein expression. In addition, the above experiments were repeated when silencing ciRS‐7, overexpressing ciRS‐7, and upregulating miR‐26a‐5p. HMEC‐1 cells formed tube‐like structures over time. Silencing ciRS‐7 suppressed viability, migration, and tube formation but promoted apoptosis. Oppositely, overexpressing ciRS‐7 reversed the effect in HMEC‐1 cells. miR‐26a‐5p expression was elevated by silencing ciRS‐7 and reduced by overexpressing ciRS‐7. Moreover, overexpressing ciRS‐7 facilitated viability, migration, and tube formation via upregulating miR‐26a‐5p. Conclusively, overexpressing ciRS‐7 mobilized phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway and suppressed c‐Jun N‐terminal kinase (JNK)/p38 pathway. ciRS‐7 exerted influence on apoptosis, viability, migration, and tube formation through mediating PI3K/AKT and JNK/p38 pathways by miR‐26a‐5p downregulation in HMEC‐1 cells. 相似文献
Phellodendron chinense Schneid is an important Chinese herb with berberine and phellodendrine in stems and leaves, but with little information available on in vitro culture of this species. Disinfection of explants in 75% alcohol for 45 s, sterilization in 0.1% HgCl2 for 20 min, and submersion in 1.0 mol L−1 gibberellin3 (GA3) solution for 24 h was the optimal condition for seed germination. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L−1 6-benzylaminopurine (6-BA) in combination with 1.5 mg L−1 1-naphthylacetic acid (NAA) was optimal for callus induction. MS medium supplemented with 2.0 mg L−1 6-BA was the appropriate medium for induction of adventitious shoots, and 1/2MS medium supplemented with 2.0 mg L−1 indole-3-butytric acid (IBA) and 0.5% active carbon was the optimal medium for root induction. The 15-d survival rate of regenerated plantlets after transplanting to basins containing perlite and peat moss (1:4) was greater than 80%, and the berberine and phellodendrine accumulation was lower in callus compared with regenerated plantlets. The establishment of highly efficient regeneration system provides technical support for genetic breeding of Phellodendron chinense Schneid.
The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa. 相似文献