Carotenoids are the pigment substance of yellow-fleshed kiwifruit, among which β-cryptoxanthin was only detected in the brighter yellow-fleshed variety “Jinshi 1”. β-carotene hydroxylase (BCH) catalyzes the formation of β-cryptoxanthin and zeaxanthin, but its molecular characteristics and functions have not been fully explained. Here, we isolated two β-carotene hydroxylase genes, AcBCH1 and AcBCH2 from kiwifruit (Actinidia chinensis), and their relative expression levels exhibited a close correlation with the content of β-cryptoxanthin. AcBCH1 catalyzed the formation of β-cryptoxanthin when transformed into the β-carotene-accumulating yeast cell. Moreover, silenced expression of AcBCH1 in kiwifruit caused a decrease in the contents of zeaxanthin, lutein and β-cryptoxanthin, and an increase in β-carotene content. The content of β-carotene decreased significantly after AcBCH1/2 were overexpressed in tomato. The content of zeaxanthin increased and β-carotene decreased in transgenic kiwifruit seedlings. The results will enrich our knowledge of the molecular mechanisms of carotenoids biosynthesis in kiwifruit.
An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine the role this mutation in rpoC plays in osmotic tolerance, we reconstructed the mutation in BW25113, and found it to confer improved tolerance to hyperosmotic stress. Metabolite analysis, exogenous supplementation assays, and cell membrane damage analysis suggest that the mechanism of improved osmotic tolerance by this rpoC mutation may be related to the higher production of acetic acid and amino acids such as proline, and increased membrane integrity in the presence of NaCl stress in exponential phase cells. Transcriptional analysis led to the findings that the overexpression of methionine related genes metK and mmuP improves osmotic tolerance in BW25113. Furthermore, deletion of a stress related gene bolA was found to confer enhanced osmotic tolerance in BW25113 and MG1655. These findings expand our current understanding of osmotic tolerance in E. coli, and have the potential to expand the utilization of high saline feedstocks and water sources in microbial fermentation.
The use of seawater in fermentation can potentially reduce the freshwater burden in the bio-based production of chemicals and fuels. We previously developed a Saccharomyces cerevisiae carotenoids hyperproducer SM14 capable of accumulating 18 mg g−1 DCW (DCW: dry cell weight) of β-carotene in rich media (YPD). In this work, the impacts of seawater on the carotenoid production of SM14 were investigated. When using nutrient-reduced media (0.1× YNB) in freshwater the β-carotene production of SM14 was 6.51 ± 0.37 mg g−1 DCW; however in synthetic seawater, the production was increased to 8.67 ± 0.62 mg g−1 DCW. We found that this improvement was partially due to the NaCl present in the synthetic seawater, since supplementation of 0.5 M NaCl in freshwater increased β-carotene production to 11.85 ± 0.77 mg g−1 DCW. The combination of synthetic seawater with higher carbon-to-nitrogen ratio (C:N = 50) further improved the β-carotene production to 10.44 ± 0.35 mg g−1 DCW. We further showed that the carotenoid production improvement in these conditions is related with lipid content and composition. These results demonstrated the benefit of using seawater to improve the production of carotenoids in S. cerevisiae, and have the potential to expand the utilization of seawater.
Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes ACT1, ACT2, GAPDH, 18S rRNA, UBQ, TUB and CYP were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. GAPDH, ACT1 and ACT2 were selected as reference genes for different genotypes of kiwifruit. GAPDH and UBQ were the best combinations of reference genes for root, stem, leaf, flower and fruit. GAPDH and ACT1 could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of ACT1 and UBQ constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit.
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