Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheles...Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.展开更多
【目的】对不同浓度磷、钾处理下小麦苗期氮养分效率相关性状进行QTL分析,以深入理解磷、钾与氮养分效率的相互关系,为氮营养相关性状的图位克隆及分子标记辅助选择育种奠定基础。【方法】采用苗期液培试验,以"川35050×山农48...【目的】对不同浓度磷、钾处理下小麦苗期氮养分效率相关性状进行QTL分析,以深入理解磷、钾与氮养分效率的相互关系,为氮营养相关性状的图位克隆及分子标记辅助选择育种奠定基础。【方法】采用苗期液培试验,以"川35050×山农483"组合衍生的小麦重组自交系群体(131个株系)为研究材料,设置了中磷中钾(MPMK)、高磷(HP)、低磷1(LP1)、低磷2(LP2)、低磷3(LP3),高钾(HK)、低钾1(LK1)、低钾2(LK2)、低钾3(LK3)共9个处理,对不同磷、钾处理下的氮养分效率相关性状进行研究,并结合分子标记遗传图谱,从整个基因组水平对与小麦苗期氮养分效率相关的10个性状进行QTL定位及遗传分析。【结果】不同处理下的10个性状共检测到137个QTL,位于除3D外的20条染色体上,大部分QTL(89.05%)仅在单一处理下被定位到,有3个QTL(QRnue-1A.2、QSnue-1A.1和QTnue-1A.1)可在至少4个处理中被检测到,有5个QTL(QRnue-1A.1、QTnue-1A.1、QSnc-4A、QRnc-6A.3和QSnue-6B)可同时在低磷和低钾环境中被检测到。本研究还检测到至少包含3个以上QTL的QTL簇17个,分别位于1A、1B、2B、2D、3A、3B、4A、4B、5D、6A、6 B、6 D和7 A染色体上,共涉及6 6个Q T L,占Q T L总数的4 8.1 8%。其中,有5个Q T L簇仅与特定磷、钾处理有关,大多数QTL簇均同时定位了不同磷、钾处理的不同性状,许多QTL簇位点还与前人定位的生物量、产量及其他养分有关。【结论】磷、钾的供应能够显著影响小麦苗期对氮素的吸收利用及其相关QTL的表达。影响苗期小麦氮养分效率相关性状的QTL大多数仅在特定处理下被检测到,但大多数QTL会形成QTL簇,构成了控制氮养分效率的QTL热点,许多热点区域也与前人定位的许多成株期性状如生物量、产量及其他养分效率有关,这些QTL/基因密集区域及其特点的发现,为我们深入理解小麦氮养分效率的遗传控制特点及其与磷、钾养分供应的关展开更多
基金sponsored by grants from the National Natural Science Foundation of China(No.82030117)the Wenzhou major scientific and technological innovation project(No.ZY2022001).
文摘Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.
文摘【目的】对不同浓度磷、钾处理下小麦苗期氮养分效率相关性状进行QTL分析,以深入理解磷、钾与氮养分效率的相互关系,为氮营养相关性状的图位克隆及分子标记辅助选择育种奠定基础。【方法】采用苗期液培试验,以"川35050×山农483"组合衍生的小麦重组自交系群体(131个株系)为研究材料,设置了中磷中钾(MPMK)、高磷(HP)、低磷1(LP1)、低磷2(LP2)、低磷3(LP3),高钾(HK)、低钾1(LK1)、低钾2(LK2)、低钾3(LK3)共9个处理,对不同磷、钾处理下的氮养分效率相关性状进行研究,并结合分子标记遗传图谱,从整个基因组水平对与小麦苗期氮养分效率相关的10个性状进行QTL定位及遗传分析。【结果】不同处理下的10个性状共检测到137个QTL,位于除3D外的20条染色体上,大部分QTL(89.05%)仅在单一处理下被定位到,有3个QTL(QRnue-1A.2、QSnue-1A.1和QTnue-1A.1)可在至少4个处理中被检测到,有5个QTL(QRnue-1A.1、QTnue-1A.1、QSnc-4A、QRnc-6A.3和QSnue-6B)可同时在低磷和低钾环境中被检测到。本研究还检测到至少包含3个以上QTL的QTL簇17个,分别位于1A、1B、2B、2D、3A、3B、4A、4B、5D、6A、6 B、6 D和7 A染色体上,共涉及6 6个Q T L,占Q T L总数的4 8.1 8%。其中,有5个Q T L簇仅与特定磷、钾处理有关,大多数QTL簇均同时定位了不同磷、钾处理的不同性状,许多QTL簇位点还与前人定位的生物量、产量及其他养分有关。【结论】磷、钾的供应能够显著影响小麦苗期对氮素的吸收利用及其相关QTL的表达。影响苗期小麦氮养分效率相关性状的QTL大多数仅在特定处理下被检测到,但大多数QTL会形成QTL簇,构成了控制氮养分效率的QTL热点,许多热点区域也与前人定位的许多成株期性状如生物量、产量及其他养分效率有关,这些QTL/基因密集区域及其特点的发现,为我们深入理解小麦氮养分效率的遗传控制特点及其与磷、钾养分供应的关
