Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and...Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.展开更多
Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils,a key limiting factor for sustainable development in modern agriculture.The polyphosphate kinase(ppk)gene-express...Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils,a key limiting factor for sustainable development in modern agriculture.The polyphosphate kinase(ppk)gene-expressing transgenic rice with a single-copy line(ETRS)is constructed to improve phosphate fertilizer utilization efficiency for phosphorus resource conservation.To investigate the potential mechanisms of the increased biomass in ETRS in low phosphate culture,ETRS was cultivated in a low inorganic phosphate(Pi)culture medium(15μmol/L Pi,LP)and a normal Pi culture medium(300μmol/L Pi,CP),respectively.After 89 d of cultivation in different concentrations of phosphate culture media,the total phosphorus,polyphosphate(polyP),biomass,photosynthetic rate,nonstructural carbohydrate(NSC)contents,related enzyme activities,and related gene expression levels were analyzed.The results showed that ETRS had a high polyP amount to promote the photosynthetic rate in LP,and its biomass was almost the same as the wild type(WT)in CP.The NSC content of ETRS in LP was higher than that of WT in LP,but slightly lower than that of WT in CP.PolyP notably promoted the sucrose phosphate synthase activities of ETRS and significantly down-regulated the expression levels of sucrose transporter genes(OsSUT3 and OsSUT4),resulting in inhibiting the transport of sucrose from shoot to root in ETRS.It was concluded that polyP can stimulate the synthesis of NSCs in LP,which improved the growth of ETRS and triggered the biological activities of ETRS to save phosphate fertilizer.Our study provides a new way to improve the utilization rate of phosphate fertilizer in rice production.展开更多
【目的】克隆甘蔗B家族SofSPSB基因并进行原核表达,为进一步研究甘蔗SPS酶学特性及SPS活性调控机制奠定基础。【方法】在进化分析的基础上,通过同源克隆获得甘蔗SofSPSB基因部分序列,再结合RACE技术获得全长cDNA序列。扩增SofSPSB基因OR...【目的】克隆甘蔗B家族SofSPSB基因并进行原核表达,为进一步研究甘蔗SPS酶学特性及SPS活性调控机制奠定基础。【方法】在进化分析的基础上,通过同源克隆获得甘蔗SofSPSB基因部分序列,再结合RACE技术获得全长cDNA序列。扩增SofSPSB基因ORF并连到原核表达载体pETBlue-2上,导入大肠杆菌BL21(DE3)中表达。【结果】通过比对B家族中进化关系很近的玉米(Zea mays)ZmSPS1和水稻(Oryza sativa)OsSPS1基因序列,并在保守区设计一对引物扩增获得甘蔗B家族SPS基因(SofSPSB)2330bp序列。结合5'-RACE和3'-RACE技术获得3481 bp SofSPSB基因全长cDNA序列,该序列包含一个3225bp的开放阅读框(ORF);起始密码子(ATG)位于转录起始位点后56bp处,终止密码子(TGA)后有一段201bp的非编码序列,并带有真核生物典型的polyA尾巴;编码1074个氨基酸,SofSPSB与Zm-SPS1、OsSPS1的核苷酸序列同源性分别为94.7%和81.3%,氨基酸序列同源性分别为96.0%和83.9%;其理论分子量Mw=118.96kDa,等电点pI=6.30。经原核表达后纯化获得带6×His标签的融合蛋白。【结论】克隆获得甘蔗B家族Sof-SPSB基因全长cDNA序列,成功构建了SofSPSB基因原核表达载体,使其在大肠杆菌BL21(DE3)中表达。展开更多
基金supported by the National Natural Science Foundation of China (32172521)the Excellent Youth Science Foundation of Heilongjiang Province,China (YQ2023C006)+1 种基金the Talent Introduction Program of Northeast Agricultural University of Chinathe Collaborative Innovation System of the Agricultural Bio-economy in Heilongjiang Province,China
文摘Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.
基金supported by the National Natural Science Foundation of China(Grant No.41871082)the Scientific Research Project of Ecological Environment Department of Jiangsu Province,China(Grant Nos.2020019 and 2021005)the National Special Program of Water Environment,China(Grant No.2017ZX07204002).
文摘Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils,a key limiting factor for sustainable development in modern agriculture.The polyphosphate kinase(ppk)gene-expressing transgenic rice with a single-copy line(ETRS)is constructed to improve phosphate fertilizer utilization efficiency for phosphorus resource conservation.To investigate the potential mechanisms of the increased biomass in ETRS in low phosphate culture,ETRS was cultivated in a low inorganic phosphate(Pi)culture medium(15μmol/L Pi,LP)and a normal Pi culture medium(300μmol/L Pi,CP),respectively.After 89 d of cultivation in different concentrations of phosphate culture media,the total phosphorus,polyphosphate(polyP),biomass,photosynthetic rate,nonstructural carbohydrate(NSC)contents,related enzyme activities,and related gene expression levels were analyzed.The results showed that ETRS had a high polyP amount to promote the photosynthetic rate in LP,and its biomass was almost the same as the wild type(WT)in CP.The NSC content of ETRS in LP was higher than that of WT in LP,but slightly lower than that of WT in CP.PolyP notably promoted the sucrose phosphate synthase activities of ETRS and significantly down-regulated the expression levels of sucrose transporter genes(OsSUT3 and OsSUT4),resulting in inhibiting the transport of sucrose from shoot to root in ETRS.It was concluded that polyP can stimulate the synthesis of NSCs in LP,which improved the growth of ETRS and triggered the biological activities of ETRS to save phosphate fertilizer.Our study provides a new way to improve the utilization rate of phosphate fertilizer in rice production.
文摘【目的】克隆甘蔗B家族SofSPSB基因并进行原核表达,为进一步研究甘蔗SPS酶学特性及SPS活性调控机制奠定基础。【方法】在进化分析的基础上,通过同源克隆获得甘蔗SofSPSB基因部分序列,再结合RACE技术获得全长cDNA序列。扩增SofSPSB基因ORF并连到原核表达载体pETBlue-2上,导入大肠杆菌BL21(DE3)中表达。【结果】通过比对B家族中进化关系很近的玉米(Zea mays)ZmSPS1和水稻(Oryza sativa)OsSPS1基因序列,并在保守区设计一对引物扩增获得甘蔗B家族SPS基因(SofSPSB)2330bp序列。结合5'-RACE和3'-RACE技术获得3481 bp SofSPSB基因全长cDNA序列,该序列包含一个3225bp的开放阅读框(ORF);起始密码子(ATG)位于转录起始位点后56bp处,终止密码子(TGA)后有一段201bp的非编码序列,并带有真核生物典型的polyA尾巴;编码1074个氨基酸,SofSPSB与Zm-SPS1、OsSPS1的核苷酸序列同源性分别为94.7%和81.3%,氨基酸序列同源性分别为96.0%和83.9%;其理论分子量Mw=118.96kDa,等电点pI=6.30。经原核表达后纯化获得带6×His标签的融合蛋白。【结论】克隆获得甘蔗B家族Sof-SPSB基因全长cDNA序列,成功构建了SofSPSB基因原核表达载体,使其在大肠杆菌BL21(DE3)中表达。
