景宁木兰热胁迫下实时荧光定量PCR内参基因的筛选

Reference genes for quantitative PCR in Magnolia sinostellata with heat stress

高温对景宁木兰Magnolia sinostellata生长产生严重影响。通过筛选景宁木兰热胁迫下稳定表达的内参基因,可以为景宁木兰在高温条件下基因表达研究提供准确数据,达到对目的基因进行准确量化的目的。以热胁迫下景宁木兰1年生实生苗的根、茎、叶组织为材料,从转录组数据库中选取13个管家基因ACTIN-7(肌动蛋白基因-7),CYP(亲环蛋白基因),EF-1α(延伸因子-1α蛋白基因),GAPDH(甘油醛-3-磷酸脱氢酶基因),GTB(GTP结合蛋白基因),NAC(NAC域蛋白基因),NADP(异柠檬酸脱氢酶基因),TEF(翻译延伸因子基因),UBC(泛素化酶基因),UBQ(多聚泛素蛋白基因),α-TUB(α微管蛋白基因),β-TUB(β微管蛋白基因)和18S(18S核糖体RNA),通过Primer 5.0进行引物设计,琼脂糖电泳和溶解曲线分析验证引物特异性;利用实时荧光定量聚合酶链式反应(qRT-PCR)技术和geNorm,NormFinder和BestKeeper等3个内参分析软件研究候选内参基因在热胁迫下不同组织中的表达稳定性,并通过CSLD3和KOR 2个目的基因的表达分析验证其可靠性。13个内参基因的电泳结果均显示单一条带,溶解曲线也均显示单一峰,证明引物特异性良好;13对引物的扩增效率均在100%左右;内参软件综合分析得到,UBC,EF-1α和ACTIN-7是景宁木兰在热胁迫下较为稳定的内参基因,以3个单独的内参基因以及内参组合(EF-1α和ACTIN-7)为参照对CSLD3和KOR的表达模式进行校准,也显示了一致的表达水平。通过qRT-PCR技术和内参软件分析,UBC,EF-1α和ACTIN-7等3个内参基因在景宁木兰热胁迫下不同组织中稳定性较高,CSLD3和KOR等2个目的基因的表达也证实了其可靠性。因此,UBC,EF-1α和ACTIN-7可以作为景宁木兰热胁迫下稳定表达的内参基因。

In field investigation high temperature was found to have a serious effect on growth of Magnolia sinostellata,an endemic endangered species in Zhejiang.To determine if screening stable reference genes of M.sinostellata with heat stress could provide accurate data for research in gene expression and achieve accurate quantification of target genes,roots,stems,and leaf tissues from annual seedlings of M.sinostellata with heat stress were taken as materials.Expression stability of 13 reference genes from transcriptome data,such as ACTIN-7(ACTIN-7),CYP(Cyclophilin),EF-1α(Elongation factors-1α),GAPDH(Glyceraldehyde-3-phosphate dehydrogenase),GBP(GTP binding protein),NAC(NAC domain protein),NADP(NADP-isocitrate dehydrogenase),TEF(Translation elongation factors),UBC(Ubiquitin-conjugating enzyme),UBQ(Polyubiquitin protein),α-TUB(Tubulin alpha),β-TUB(Tubulin beta),and 18S(18S ribosomal RNA)were detected and evaluated by quantitative reverse transcriptase(qRT)-polymerase chain reaction(PCR)along with geNorm,NormFinder,and BestKeeper.Also,two target genes,CSLD3 and KOR,were analyzed to verify reliability of the screened reference genes.Specificity of the 13 pairs of primers was validated with agarose gel electrophoresis and a melting curve.Results showed that the 13 reference genes had a distinct PCR product in the electrophoresis figure,and the melting curve showed a single peak.Amplification efficiency of the 13 pairs of primers was about 100%with UBC,EF-1α,and ACTIN-7 being stable reference genes in different tissues of M.sinostellata.Additionally,the two target genes,CSLD3 and KOR,showed consistent expression profiles when normalized by UBC,EF-1α,and ACTIN-7,as well as the combination(EF-1αand ACTIN-7).UBC,EF-1α,and ACTIN-7 were the best choices in different tissues of M.sinostellata with heat stress,and since expression analysis of the target genes,CSLD3 and KOR,further confirmed reliability of these genes,UBC,EF-1α,and ACTIN-7 could serve as references genes for expression among different tissues in M.sinostellata with heat stress.