Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cott...Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.展开更多
A gene （NANOC-D6D） encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata （Droop） D. J. Hibberd （Eustigmatophyceae）...A gene （NANOC-D6D） encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata （Droop） D. J. Hibberd （Eustigmatophyceae）. The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids （LCPUFAs）, including eicosapentaenoic acid （20：5n-3, EPA）. The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NAN OC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid （LA） and a-linolenic acid （ALA）.展开更多
Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of ...Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.展开更多
A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading...A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.展开更多
Fatty acid(FA)desaturases,as the key enzymes in lipid metabolism,are responsible for biosynthesis of the unsaturated fatty FAs,which play important roles in maintaining cell membrane integrity and multiple stress resp...Fatty acid(FA)desaturases,as the key enzymes in lipid metabolism,are responsible for biosynthesis of the unsaturated fatty FAs,which play important roles in maintaining cell membrane integrity and multiple stress responses.Although attention has been drawn to some plant FA desaturase genes,their global landscape in oil crops is still lacking.Here,we performed systematic characterization and phylogenomic synteny network analyses of the FA desaturase gene family in polyploid oil crop B.napus and other 54 species covering major streptophyte lineages.A total of 1653 FA desaturase genes were identified from these plant genomes.Based on the broad-scale family phylogeny and functional domains,we proposed a unified eight-group classification system for angiosperm FA desaturases,and found that the origin of genes responsible for FA desaturation evolved early and some genes were absent in different species.Phylogenomic analyses revealed deeply conserved syntenic relationships within each of the eight FA desaturase groups.B.napus contains up to 93 FA desaturase genes from the eight groups.Recurrent duplication events in Brassicaceae contributed to the expansion of FA desaturase genes in B.napus,leading to further functional diversification.These FA desaturase genes exhibited spatio-temporal specific expression patterns in different tissues of B.napus,and a set of FA desaturase genes seem to be orchestrated by key transcriptional factors during seed development,such as zf-HD,B3,GATA3,PEI1,NFYA7,YAB1 and YAB2.Altogether,our data have inferred the evolutionary trajectory of this important gene family across distinct plant lineages,providing theoretical basis for future manipulation of FA desaturase genes to improve the seed oil quality of B.napus.展开更多
To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The res...To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.展开更多
ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(...ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.展开更多
文摘Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.
基金Supported by the National Key Technology R&D Program in the 11th Five-Year Plan of China (No. 2006BAD09A03-2)the National High-tech Research and Development Program of China(863 Program) (No. 2007AA09Z427)
文摘A gene （NANOC-D6D） encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata （Droop） D. J. Hibberd （Eustigmatophyceae）. The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids （LCPUFAs）, including eicosapentaenoic acid （20：5n-3, EPA）. The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NAN OC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid （LA） and a-linolenic acid （ALA）.
基金Project supported by"Tenth Five Years"Key Program of the State Science and Technology Commission in China(Grant Nos.2002BA901A15,2004BA411B01)
文摘Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.
文摘A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.
基金funded by the Agricultural Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences(CAAS-ASTIP-OCRI),the National Natural Science Foundation of China(grant number 31801399)the Research Foundation of Education Bureau of Hunan Province,China(grant number 21A0135)。
文摘Fatty acid(FA)desaturases,as the key enzymes in lipid metabolism,are responsible for biosynthesis of the unsaturated fatty FAs,which play important roles in maintaining cell membrane integrity and multiple stress responses.Although attention has been drawn to some plant FA desaturase genes,their global landscape in oil crops is still lacking.Here,we performed systematic characterization and phylogenomic synteny network analyses of the FA desaturase gene family in polyploid oil crop B.napus and other 54 species covering major streptophyte lineages.A total of 1653 FA desaturase genes were identified from these plant genomes.Based on the broad-scale family phylogeny and functional domains,we proposed a unified eight-group classification system for angiosperm FA desaturases,and found that the origin of genes responsible for FA desaturation evolved early and some genes were absent in different species.Phylogenomic analyses revealed deeply conserved syntenic relationships within each of the eight FA desaturase groups.B.napus contains up to 93 FA desaturase genes from the eight groups.Recurrent duplication events in Brassicaceae contributed to the expansion of FA desaturase genes in B.napus,leading to further functional diversification.These FA desaturase genes exhibited spatio-temporal specific expression patterns in different tissues of B.napus,and a set of FA desaturase genes seem to be orchestrated by key transcriptional factors during seed development,such as zf-HD,B3,GATA3,PEI1,NFYA7,YAB1 and YAB2.Altogether,our data have inferred the evolutionary trajectory of this important gene family across distinct plant lineages,providing theoretical basis for future manipulation of FA desaturase genes to improve the seed oil quality of B.napus.
基金Thanks for the technical support of Dr.Daqi Fu and Dr.Lanhuan Meng of China Agricultural University.This work was supported by Beijing Municipal Science and Technology Project(Grant No.Z181100002418006)the Fundamental Research Fund for the Central University(Grant No.2015ZCQ-YL-03)the World-Class Discipline Construction and Characteristic Development Guidance Funds for Beijing Forestry University(Grant No.2019XKJS0323).
文摘To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.
基金supported by National Natural Science Foundation of China(31500247)Key Project of Department of Education of Sichuan Province(14ZA0016)Natural Science Foundation of Zhejiang Province(LZ15C150001)
文摘ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.
