The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization....The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.展开更多
Three Gracilaria species,G.chouae,G.blodgettii,G.vermiculophylla and a close relative species,Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis,are the typically indigenous species which ...Three Gracilaria species,G.chouae,G.blodgettii,G.vermiculophylla and a close relative species,Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis,are the typically indigenous species which are important resources for the production of special proteins,phycobilisomes,special carbohydrates,and agar in China.In this study,de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time.Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G.lemaneiformis and other three Gracilaria species.Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species.The genes encoding allophycocyanin,phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G.lemaneiformis had close relationship to genus Gracilaria,as well as that within genus Gracilaria,G.chouae had closer relationship to G.vermiculophylla rather than to G.blodgettii.The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.展开更多
基金Supported by the Key Program of Science and Technology Innovation in Ningbo (No.2019B10009)the National Natural Science Foundation of China (No.41476111)。
文摘The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.
基金The National Natural Science Foundation of China under contract Nos 31140070,31271397 and 41206116the algal transcrip-tome sequencing was supported by 1KP Project(www.onekp.com)
文摘Three Gracilaria species,G.chouae,G.blodgettii,G.vermiculophylla and a close relative species,Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis,are the typically indigenous species which are important resources for the production of special proteins,phycobilisomes,special carbohydrates,and agar in China.In this study,de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time.Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G.lemaneiformis and other three Gracilaria species.Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species.The genes encoding allophycocyanin,phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G.lemaneiformis had close relationship to genus Gracilaria,as well as that within genus Gracilaria,G.chouae had closer relationship to G.vermiculophylla rather than to G.blodgettii.The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.
