Quantitative real-time polymerase chain reaction(q RT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts(expression analysis). It is also employed for studying heterosis,...Quantitative real-time polymerase chain reaction(q RT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts(expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of q RT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea(SS), Crassostrea angulata(AA) and their hybrid(SA), which included three ribosomal genes, 28 S ribosomal protein S5(RPS5), ribosomal protein L35(RPL35), and 60 S ribosomal protein L29(RPL29); three structural genes, tubulin gamma(TUBγ), annexin A6 and A7(AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase(OD), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and glutathione S-transferase P1(GSP); two transcription factors, elongation factor 1 alpha and beta(EF1α and EF1β); and one protein synthesis gene(ubiquitin(UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, ge Norm, Norm Finder and Best Keeper, were used to evaluate the expression stability of these candidate genes. Best Keeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid(SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.展开更多
Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperatur...Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperature.Here,we used high-throughput sequencing to analyze the molecular processes in the hybrids under and after thermal stress.The hybrids were cultured in the seawater with an increasing temperature from 25℃to 40℃during 10 hours,which is regarded as the thermal stress stage.Then the temperature decreased from 40℃to 25℃within 2 h,which is regarded as the recovery stage.In this study,1293 significant diffe-rentially expressed genes(DEGs)were obtained under thermal stress,of which 576 were upregulated and 717 were downregulated,and 740 significant differentially expressed genes(DEGs)were obtained in the recovery stage,of which the number of upregulated and downregulated genes was 409 and 331,respectively.The antigen processing and presentation,NOD-like,and NF-kappa B path-ways were significantly enriched during the thermal stress stage.The MAPK and PPAR signaling pathways were significantly enrich-ed during the recovery stage.The HSP70,HSP90,and CANX genes were strongly and rapidly upregulated in the control/thermal stress groups but were slightly less upregulated in the thermal stress/recovery group.These results indicate that the innate immune system or nonspecific immunity was deployed to protect interior tissues from thermal stress.In addition,85%of the mutual DEGs were involved in bidirectional regulation(up/down or down/up)when the oysters were removed from the thermal stress to recover.This study provides preliminary insight into the molecular response of C.sikamea(♀)and C.gigas(♂)hybrids to thermal stress and provides a basis for future studies on temperature-adaptation and the possible expansion of hybrid breeding.展开更多
基金supported by the National Natural Science Foundation of China (No.31172403)
文摘Quantitative real-time polymerase chain reaction(q RT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts(expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of q RT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea(SS), Crassostrea angulata(AA) and their hybrid(SA), which included three ribosomal genes, 28 S ribosomal protein S5(RPS5), ribosomal protein L35(RPL35), and 60 S ribosomal protein L29(RPL29); three structural genes, tubulin gamma(TUBγ), annexin A6 and A7(AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase(OD), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and glutathione S-transferase P1(GSP); two transcription factors, elongation factor 1 alpha and beta(EF1α and EF1β); and one protein synthesis gene(ubiquitin(UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, ge Norm, Norm Finder and Best Keeper, were used to evaluate the expression stability of these candidate genes. Best Keeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid(SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.
基金supported by the National Natural Sci-ence Foundation of China(No.31172403).
文摘Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperature.Here,we used high-throughput sequencing to analyze the molecular processes in the hybrids under and after thermal stress.The hybrids were cultured in the seawater with an increasing temperature from 25℃to 40℃during 10 hours,which is regarded as the thermal stress stage.Then the temperature decreased from 40℃to 25℃within 2 h,which is regarded as the recovery stage.In this study,1293 significant diffe-rentially expressed genes(DEGs)were obtained under thermal stress,of which 576 were upregulated and 717 were downregulated,and 740 significant differentially expressed genes(DEGs)were obtained in the recovery stage,of which the number of upregulated and downregulated genes was 409 and 331,respectively.The antigen processing and presentation,NOD-like,and NF-kappa B path-ways were significantly enriched during the thermal stress stage.The MAPK and PPAR signaling pathways were significantly enrich-ed during the recovery stage.The HSP70,HSP90,and CANX genes were strongly and rapidly upregulated in the control/thermal stress groups but were slightly less upregulated in the thermal stress/recovery group.These results indicate that the innate immune system or nonspecific immunity was deployed to protect interior tissues from thermal stress.In addition,85%of the mutual DEGs were involved in bidirectional regulation(up/down or down/up)when the oysters were removed from the thermal stress to recover.This study provides preliminary insight into the molecular response of C.sikamea(♀)and C.gigas(♂)hybrids to thermal stress and provides a basis for future studies on temperature-adaptation and the possible expansion of hybrid breeding.
