[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone i...[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.展开更多
Rice tillering is an important agronomic trait affecting grain yield.Here,we identified a high-tillering mutant tillering20(t20),which could be restored to the wild type by treatment with the strigolactone(SL)analog r...Rice tillering is an important agronomic trait affecting grain yield.Here,we identified a high-tillering mutant tillering20(t20),which could be restored to the wild type by treatment with the strigolactone(SL)analog rac-GR24.T20 encodes a chloroplast ζ-carotene isomerase(Z-ISO),which is involved in the biosynthesis of ca-rotenoids and their metabolites,SL and abscisic acid(ABA).The t20 mutant has reduced SL and ABA,raising the question of how SL and ABA biosynthesis is coordinated,and whether they have overlapping functions in tillering.We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-p-carotene biosynthesis.Importantly,rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXY-CAROTENOID DIOXYGENASE 1(OsNCED1)through induction of Oryza sativa HOMEOBOX12(0sHOX12),promoting ABA biosynthesis in shoot base.On the other hand,ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis.ABA treatment reduced the number of basal tillers in both t20 and wild-type plants.Furthermore,while ABA-deficient mu-tants aba1 and aba2 had the same number of basal tillers as wild type,they had more unproductive upper tillers at maturity.This work demonstrates complex interactions in the biosynthesis of carotenoid,SLs and ABA,and reveals a role for ABA in the regulation of rice tillering.展开更多
Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection me...Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.展开更多
Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and...Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.展开更多
文摘[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.
基金grants from the Chinese Ministry of Science and Technology(2016YFD0100901)the Ministry of Agriculture of China(2016ZX08011001)+2 种基金the CAS President’s International Fellowship Initiative(2018VBA0025)the Youth Innovation Promotion Association CAS(2019099)the Strategic Priority Research Program of CAS(XDB27010100)(Beltran et al.,2015).
文摘Rice tillering is an important agronomic trait affecting grain yield.Here,we identified a high-tillering mutant tillering20(t20),which could be restored to the wild type by treatment with the strigolactone(SL)analog rac-GR24.T20 encodes a chloroplast ζ-carotene isomerase(Z-ISO),which is involved in the biosynthesis of ca-rotenoids and their metabolites,SL and abscisic acid(ABA).The t20 mutant has reduced SL and ABA,raising the question of how SL and ABA biosynthesis is coordinated,and whether they have overlapping functions in tillering.We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-p-carotene biosynthesis.Importantly,rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXY-CAROTENOID DIOXYGENASE 1(OsNCED1)through induction of Oryza sativa HOMEOBOX12(0sHOX12),promoting ABA biosynthesis in shoot base.On the other hand,ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis.ABA treatment reduced the number of basal tillers in both t20 and wild-type plants.Furthermore,while ABA-deficient mu-tants aba1 and aba2 had the same number of basal tillers as wild type,they had more unproductive upper tillers at maturity.This work demonstrates complex interactions in the biosynthesis of carotenoid,SLs and ABA,and reveals a role for ABA in the regulation of rice tillering.
基金supported in part by the Natural Science Foundation of Beijing, China (5121001)the Cultivate New Varieties of Genetically Modified Organisms Technology Major Projects, the Ministry of Science and Technology of China (2009ZX08012-006B)
文摘Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.
文摘Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.
