A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total ...A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total of 14 788 SSRs were observed in the whole genomic DNA sequence, about one every 2.57 kb, with the criteria of SSR length >15 bp and 80% matches. The most abundant microsatellite was trinucleotide repeat, the number was 4 729, followed by hexanucleotide and mononucleotide repeats, the numbers were 2 940 and 2 489 respectively, and the least abundance was dinucleotide repeat, only 691 were found. Among the 10 082 ORFs, 4 094 SSRs were harbored in 2 373 ORF (no intron) of the organism. One thousand and fifty six ORFs harbored only one SSR. Similar with other organisms, tri- and hexanucleotide repeats were predominant in ORFs, 54.1 and 48.8% of tri- and hexanucleotide repeats were distributed in ORF region. The density of these two motifs was overpresented in coding regions, because ORF region and coding region constitutes only 46 and 38.3% of genomic sequence, respectively. Upstream and downstream 300 bp of regulatory regions were high density regions of SSRs, particularly density of pentanucleotide SSR in upstream region was as high as five times of average density in genomic DNA, density of di- and tetranucleotide SSR was also more than two times of average density. The density of penta-, tetra-, di- and mononucleotide SSRs was relatively higher than average density. There were 47 SSRs in mitochondria 64 840 bp DNA sequence, their distribution is similar with genomic DNA sequence. These results suggested that SSRs were clustered in regulatory regions of genomic DNA.展开更多
[Objective] This study aimed to investigate the regulatory mechanisms of carotenoid biosynthesis in Neurospora crassa. [Method] Gene knockout mutants pro- ducing less carotenoid were screened from 6 087 mutants; the y...[Objective] This study aimed to investigate the regulatory mechanisms of carotenoid biosynthesis in Neurospora crassa. [Method] Gene knockout mutants pro- ducing less carotenoid were screened from 6 087 mutants; the yield of carotenoid and asexual spore was measured; finally fluorescent quantitative real-time PCR was adopted to analyze the transcription of genes related to carotenoid synthesis and asexual sporulation, [Result] Six knockout mutants produced less carotenoid. In one of them, the yield of both carotenoid and asexual spore reduced, because the gene which encodes an ATP-dependent chromatin remodelling complex ATPase chain ISW1 was knocked out. This gene was named /ca-1 in this study. And the /ca-1 deletion result- ed in a reduction of 88% in conidial production and a decrease of 81% in carotenoid production. [Conclusion] The Ica-1 positively regulates the carotenoid syn- thesis and asexual sporulation in N. crassa.展开更多
Specific-locus mutations induced in the ad-3region of two-component heterokaryons ofNeurospora have been compared by means ofMutational Spectra.Genetic characterization ofad-3 mutations has revealed that there are maj...Specific-locus mutations induced in the ad-3region of two-component heterokaryons ofNeurospora have been compared by means ofMutational Spectra.Genetic characterization ofad-3 mutations has revealed that there are majorclasses:gene/point mutations,multilocus deletionmutations and unknowns (mutants that grow toorapidly alone on minimal medium to be classified).展开更多
Vitamin A deficiency remains a severe global health issue,which creates a need to biofortify crops with provitamin A carotenoids(PACs).Expanding plant cell capacity for synthesis and storing of PACs outside the plasti...Vitamin A deficiency remains a severe global health issue,which creates a need to biofortify crops with provitamin A carotenoids(PACs).Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored.Here,we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves,Arabidopsis seeds,and citrus callus cells,using a fungal(Neurospora crassa)carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs,including β-carotene.This strategy led to the accumulation of significant amounts of phytoene and γ-and β-carotene,in addition to fungal,health-promoting carotenes with 13 conjugated double bonds,such as the PAC torulene,in the cytosol.Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production.Engineered carotenes accumulate in cytosolic lipid droplets(CLDs),which represent a novel sequestering sink for storing these pigments in plant cytosol.Importantly,β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidialβ-carotene.Moreover,engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids,including retinal,the aldehyde corresponding to vitamin A.Collectively,our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues,especially in lipid-storing seeds,and thus paves the way for further optimization of carotenoid biofortification in crops.展开更多
Genes of NADP-glutamate dehydrogenase (NADP-GDH) were cloned from Neurospora intermedia (Ni), N. crassa (Nc), and N. sitophila (Ns). The sequences showed a high degree of homology at the cDNA and protein level. The th...Genes of NADP-glutamate dehydrogenase (NADP-GDH) were cloned from Neurospora intermedia (Ni), N. crassa (Nc), and N. sitophila (Ns). The sequences showed a high degree of homology at the cDNA and protein level. The three GDH genes were cloned into pET30a and expressed in E. coli. The activity assay of purified GDH showed that the Ni-GDH had a higher activity and affinity to ammonia than Ns-GDH, and Nc-GDH. The Km value of Ni-GDH ranges from 0.3 to 0.45 nimol/L. Ni-gdh gene was transformed to Nicotiana bethamiana plants. The transformed plants grew much better in low nitrogen media than the only ROKII vector transformed control.展开更多
Circadian clocks are the internal time-keeping mechanisms for organisms to synchronize their cellular and physiological processes to the daily light/dark cycles.The molecular mechanisms underlying circadian clocks are...Circadian clocks are the internal time-keeping mechanisms for organisms to synchronize their cellular and physiological processes to the daily light/dark cycles.The molecular mechanisms underlying circadian clocks are remarkably similar in eukaryotes.Neurospora crassa,a filamentous fungus,is one of the best understood model organisms for circadian research.In recent years,accumulating data have revealed complex regulation in the Neurospora circadian clock at transcriptional,posttranscriptional,post-translational and epigenetic levels.Here we review the recent progress towards our understanding of the molecular mechanism of the Neurospora circadian oscillator.These advances have provided novel insights and furthered our understanding of the mechanism of eukaryotic circadian clocks.展开更多
Objective:To isolate and demonstrate the mechanism of metal transport in cobalt-sensitive mutant(CSM)of Neurospora crassa(N.crassa).Methods:Isolation of CSM of N.crassa,I_(50)determination,growth measurements,metal io...Objective:To isolate and demonstrate the mechanism of metal transport in cobalt-sensitive mutant(CSM)of Neurospora crassa(N.crassa).Methods:Isolation of CSM of N.crassa,I_(50)determination,growth measurements,metal ion uptake studies and sexual crosses were performed to determine the mechanism of sensitivity and locus.Results:CSMs of N.crassa were isolated by mutagenesis with diethyl sulfate.More than 500 isolates were screened and out of these isolates,CSM-I was 5-fold and CSM-II was 10-fold sensitive to Co on liquid medium as compared to the wild type.Compositional analysis of cell wall revealed the decrease in total phosphate content.N.crassa CSM bound much less cobalt to cell wall fraction than wild type.The data indicated closer linkage between resistance and mating type locus(mat),which is,located on LG I.Conclusions:A CSM of N.crassa is 5-fold more sensitive than wild type and cross sensitive to nickel and copper and hyper-accumulates 2-4 fold more toxic metal ions over wild type.The mechanism for sensitivity is decreased in cobalt-binding to cell wall fraction and increased intracellular uptake.N.crassa-acon-3 morphologically resembles the CSM,cobalt-sensitive and maps to similar locus.展开更多
基金the National Natural Science Foundation of China(30360061) Natural Science Foundation of Yunnan Province of China(1999一c0008z).
文摘A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total of 14 788 SSRs were observed in the whole genomic DNA sequence, about one every 2.57 kb, with the criteria of SSR length >15 bp and 80% matches. The most abundant microsatellite was trinucleotide repeat, the number was 4 729, followed by hexanucleotide and mononucleotide repeats, the numbers were 2 940 and 2 489 respectively, and the least abundance was dinucleotide repeat, only 691 were found. Among the 10 082 ORFs, 4 094 SSRs were harbored in 2 373 ORF (no intron) of the organism. One thousand and fifty six ORFs harbored only one SSR. Similar with other organisms, tri- and hexanucleotide repeats were predominant in ORFs, 54.1 and 48.8% of tri- and hexanucleotide repeats were distributed in ORF region. The density of these two motifs was overpresented in coding regions, because ORF region and coding region constitutes only 46 and 38.3% of genomic sequence, respectively. Upstream and downstream 300 bp of regulatory regions were high density regions of SSRs, particularly density of pentanucleotide SSR in upstream region was as high as five times of average density in genomic DNA, density of di- and tetranucleotide SSR was also more than two times of average density. The density of penta-, tetra-, di- and mononucleotide SSRs was relatively higher than average density. There were 47 SSRs in mitochondria 64 840 bp DNA sequence, their distribution is similar with genomic DNA sequence. These results suggested that SSRs were clustered in regulatory regions of genomic DNA.
基金Supported by the National Natural Science Foundation of China (31000551 30970127)~~
文摘[Objective] This study aimed to investigate the regulatory mechanisms of carotenoid biosynthesis in Neurospora crassa. [Method] Gene knockout mutants pro- ducing less carotenoid were screened from 6 087 mutants; the yield of carotenoid and asexual spore was measured; finally fluorescent quantitative real-time PCR was adopted to analyze the transcription of genes related to carotenoid synthesis and asexual sporulation, [Result] Six knockout mutants produced less carotenoid. In one of them, the yield of both carotenoid and asexual spore reduced, because the gene which encodes an ATP-dependent chromatin remodelling complex ATPase chain ISW1 was knocked out. This gene was named /ca-1 in this study. And the /ca-1 deletion result- ed in a reduction of 88% in conidial production and a decrease of 81% in carotenoid production. [Conclusion] The Ica-1 positively regulates the carotenoid syn- thesis and asexual sporulation in N. crassa.
文摘Specific-locus mutations induced in the ad-3region of two-component heterokaryons ofNeurospora have been compared by means ofMutational Spectra.Genetic characterization ofad-3 mutations has revealed that there are majorclasses:gene/point mutations,multilocus deletionmutations and unknowns (mutants that grow toorapidly alone on minimal medium to be classified).
基金supported by baseline funding and Competitive Research Grants(CRG 2017,CRG 2020)given to Salim Al-Babili from King Abdullah University of Science and Technology(KAUST).
文摘Vitamin A deficiency remains a severe global health issue,which creates a need to biofortify crops with provitamin A carotenoids(PACs).Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored.Here,we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves,Arabidopsis seeds,and citrus callus cells,using a fungal(Neurospora crassa)carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs,including β-carotene.This strategy led to the accumulation of significant amounts of phytoene and γ-and β-carotene,in addition to fungal,health-promoting carotenes with 13 conjugated double bonds,such as the PAC torulene,in the cytosol.Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production.Engineered carotenes accumulate in cytosolic lipid droplets(CLDs),which represent a novel sequestering sink for storing these pigments in plant cytosol.Importantly,β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidialβ-carotene.Moreover,engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids,including retinal,the aldehyde corresponding to vitamin A.Collectively,our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues,especially in lipid-storing seeds,and thus paves the way for further optimization of carotenoid biofortification in crops.
基金Thanks are due to Prof. Qian Shijun of the Institute of Microbiology of the CAS for valuable suggestion.
文摘Genes of NADP-glutamate dehydrogenase (NADP-GDH) were cloned from Neurospora intermedia (Ni), N. crassa (Nc), and N. sitophila (Ns). The sequences showed a high degree of homology at the cDNA and protein level. The three GDH genes were cloned into pET30a and expressed in E. coli. The activity assay of purified GDH showed that the Ni-GDH had a higher activity and affinity to ammonia than Ns-GDH, and Nc-GDH. The Km value of Ni-GDH ranges from 0.3 to 0.45 nimol/L. Ni-gdh gene was transformed to Nicotiana bethamiana plants. The transformed plants grew much better in low nitrogen media than the only ROKII vector transformed control.
基金This work was supported by grants from National Institutes of Health(Grant Nos.GM062591 and GM068496 to Y.L.)and Welch Foundation(to Y.L.).Y.L.is the Louise W.Kahn endowed scholar in Biomedical Research at University of Texas South-western Medical Center.
文摘Circadian clocks are the internal time-keeping mechanisms for organisms to synchronize their cellular and physiological processes to the daily light/dark cycles.The molecular mechanisms underlying circadian clocks are remarkably similar in eukaryotes.Neurospora crassa,a filamentous fungus,is one of the best understood model organisms for circadian research.In recent years,accumulating data have revealed complex regulation in the Neurospora circadian clock at transcriptional,posttranscriptional,post-translational and epigenetic levels.Here we review the recent progress towards our understanding of the molecular mechanism of the Neurospora circadian oscillator.These advances have provided novel insights and furthered our understanding of the mechanism of eukaryotic circadian clocks.
文摘Objective:To isolate and demonstrate the mechanism of metal transport in cobalt-sensitive mutant(CSM)of Neurospora crassa(N.crassa).Methods:Isolation of CSM of N.crassa,I_(50)determination,growth measurements,metal ion uptake studies and sexual crosses were performed to determine the mechanism of sensitivity and locus.Results:CSMs of N.crassa were isolated by mutagenesis with diethyl sulfate.More than 500 isolates were screened and out of these isolates,CSM-I was 5-fold and CSM-II was 10-fold sensitive to Co on liquid medium as compared to the wild type.Compositional analysis of cell wall revealed the decrease in total phosphate content.N.crassa CSM bound much less cobalt to cell wall fraction than wild type.The data indicated closer linkage between resistance and mating type locus(mat),which is,located on LG I.Conclusions:A CSM of N.crassa is 5-fold more sensitive than wild type and cross sensitive to nickel and copper and hyper-accumulates 2-4 fold more toxic metal ions over wild type.The mechanism for sensitivity is decreased in cobalt-binding to cell wall fraction and increased intracellular uptake.N.crassa-acon-3 morphologically resembles the CSM,cobalt-sensitive and maps to similar locus.
