摘要
为探究4个内参基因在草鱼脂肪组织和脂肪细胞中的稳定表达情况及筛选稳定性最佳的内参基因,采用实时荧光定量PCR(qRT-PCR)技术检测了在水温26.5~27.5℃下,摄食脂肪含量为4%、8%和12%的饲料4周、体质量为(15.0±0.5)g的草鱼腹腔脂肪组织中及在成脂培养基中诱导分化至第6天时,用含0、40、80、120 mmol/L的油酸处理的草鱼脂肪细胞中的18S rRNA基因、β-actin基因、GAPDH基因和EF1α基因等4个候选内参基因的转录水平,随后采用GeNorm、NormFinder和Bestkeeper软件分析其表达稳定性,以筛选出特定条件下草鱼脂肪组织及脂肪细胞中稳定表达的内参基因或其组合。结果显示,候选内参基因的定量PCR引物均可获得特异性扩增产物和理想的扩增效率。在草鱼脂肪组织和脂肪细胞中,候选内参基因表达稳定性顺序分别为18S rRNA>β-actin>GAPDH>EF1α和18S rRNA=β-actin>GAPDH>EF1α。GeNorm标准化因子配对差异分析显示,其配对变异值均小于0.15,即草鱼脂肪组织及脂肪细胞中最适内参基因数至少有2个。结果表明,在草鱼脂肪组织和脂肪细胞中稳定表达的是18S rRNA基因,建议选择18S rRNA、β-actin基因作为草鱼脂肪组织及脂肪细胞内参基因组合。
To explore the stable expression of the four reference genes in grass carp Ctenopharyngodon idellus adipose tissue and adipocytes,and to select the best stable reference genes,transcription levels of 4 candidate reference genes including 18S rRNA,β-actin,GAPDH and EF1αwere investigated in abdominal adipose tissue of grass carp with body weight of(15.0±0.5)g fed diets containing 4%,8%and 12%of lipid at water temperature of 26.5-27.5℃for 4 weeks when the adipocytes were treated with oleic acid concentrations of 0,40,80 and 120 mmol/L by real-time fluorescence quantitative PCR(qRT-PCR),and then the stability of their expression levels were analyzed by software GeNorm,NormFinder and BestKeeper to screen out the reference genes or combinations stably expressed in adipose tissue and adipose cells.The results showed that specific amplification products and ideal amplification efficiency were obtained by all the quantitative PCR primers for candidate reference genes.The descending order of expression stability for candidate reference genes was expressed as 18S rRNA>β-actin>GAPDH>EF1αand 18S rRNA=β-actin>GAPDH>EF1αIn in adipose tissue and adipocytes.The differentiation analysis of standardized factor pairing of GeNorm showed that its V n/V(n+1)was found to be less than 0.15,that is,with the optimal reference genes number of at least 2 in adipose tissue and adipocytes.The findings indicated that the reference gene stably expressed in adipose tissue and adipocytes of grass carp was 18S rRNA.It was suggested that 18S rRNA andβ-actin should be selected as the most suitable reference gene combination in adipose tissue and cells.
作者
胡泽超
邹孝翠
孙健
边晨晨
吉红
HU Zechao;ZOU Xiaocui;SUN Jian;BIAN Chenchen;JI Hong(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China)
出处
《水产科学》
CAS
CSCD
北大核心
2023年第4期547-555,共9页
Fisheries Science
基金
国家自然科学基金面上项目(31772863).
关键词
实时荧光定量PCR
草鱼
内参基因筛选
脂肪组织
脂肪细胞
表达稳定性
real-time quantitative PCR
Ctenopharyngodon idellus
screening of reference gene
adipose tissue
adipocyte
expression stability
