The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected fiv...The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.展开更多
Aquaculture in saline-alkaline water has a major problem： microalgal blooming causes the pH of water to increase dramatically, thereby causing damage to the reared organisms. To solve this problem, we set out to find...Aquaculture in saline-alkaline water has a major problem： microalgal blooming causes the pH of water to increase dramatically, thereby causing damage to the reared organisms. To solve this problem, we set out to find a candidate filter-feeding bivalve species suitable for saline-alkaline water to graze on microalgae and to control the pH. In the current study, we investigated the effect of carbonate alkalinity （CA, 2.5, 10.0, and 20.0 meq/L） and pH （8.0, 8.5, and 9.0） on the grazing capacity （GC） of the clam Cyclina sinensis. Additionally, the effect of clam size （small, medium, and large） and microalgae species （Nannochloropsis oculata, Chaetoceros miielleri, and lsochrysis galbana）, and the effect of bottom sediment characteristic （mud, sandy mud, and muddy sand） and thickness （3 and 6 cm） were analyzed as well. The results show that the GC on L galbana was the highest and small size had the maximum GC/W （W： wet weight including body and shells）. No significant differences were observed between sediment type and thickness. Regarding CA and pH, a significant decrease in GC by the pH or by their interaction was found. The GC ofC. sinensis was not greatly reduced in the treatments ofpH≤8.5 and CA≤20.0, and also not affected by bottom sediment type, indicating that this clam is capable to manage microalgal concentrations and might be a candidate species for pH reduction in saline-alkaline water ponds.展开更多
基金Supported by the funding for school-level research projects of Yancheng Institute of Technology(No.xjr2019047)the National Natural Science Foundation of China(No.31902362)。
文摘The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.
基金Supported by the Special Fund of Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes titled“Eff ect of p H on the Larva Development and Carbon Sequestration of Several Intertidal Zone Bivalves in the East China Sea(Chinese Academy of Fishery Sciences)(No.2014A01YY02)the Returned Central Royalties for Using Sea Areas titled“Demonstration for Ecological Restoration and Environmental Improvement in Fengxian Sea Area Located in the Northern Seacoast of Hangzhou Bay”
文摘Aquaculture in saline-alkaline water has a major problem： microalgal blooming causes the pH of water to increase dramatically, thereby causing damage to the reared organisms. To solve this problem, we set out to find a candidate filter-feeding bivalve species suitable for saline-alkaline water to graze on microalgae and to control the pH. In the current study, we investigated the effect of carbonate alkalinity （CA, 2.5, 10.0, and 20.0 meq/L） and pH （8.0, 8.5, and 9.0） on the grazing capacity （GC） of the clam Cyclina sinensis. Additionally, the effect of clam size （small, medium, and large） and microalgae species （Nannochloropsis oculata, Chaetoceros miielleri, and lsochrysis galbana）, and the effect of bottom sediment characteristic （mud, sandy mud, and muddy sand） and thickness （3 and 6 cm） were analyzed as well. The results show that the GC on L galbana was the highest and small size had the maximum GC/W （W： wet weight including body and shells）. No significant differences were observed between sediment type and thickness. Regarding CA and pH, a significant decrease in GC by the pH or by their interaction was found. The GC ofC. sinensis was not greatly reduced in the treatments ofpH≤8.5 and CA≤20.0, and also not affected by bottom sediment type, indicating that this clam is capable to manage microalgal concentrations and might be a candidate species for pH reduction in saline-alkaline water ponds.
