The Selection Of Reference Genes In Saccharina Japonica And Phylogenetic Analysis Of Partial Housekeeping Genes

qPCR is the most accurate and available method to measure gene expressionlevel,in recent years this technology become more and more important to study thechange of functional gene expression.When we study the expression of gene byqPCR,we need reference genes to calibrate the experimental course.In this situation，selection of an appropriate reference gene is very important for an accurate result.Wehave selected8candidate reference genes from lots of house keeping genes，they areAct(Actin)，EF1α（Translation elongation factor1alpha），UBA80（Ubiquitin80），GAPDH（glyceraldehyde-3-phosphate dehydrogenase），UBA52（Ubiquitin52），18S（18S rRNA），TUA（Alpha tubulin）and TUB（Beta tubulin）.These house-keepinggenes are very ancient, and is highly diversed in many organisms.Except for the sequences annotated in large-scale sequencing of Ectocarpussiliculosus, there is limited reseach on the cloning of these housekeeping genes.In ourstudy, we have cloned7genes above,except18S.It is the first time that obtaining thesequences of these genes in Phaeophyta.The length of these genes are387~1362bpand the GC content are59.4%~65.4%.These genes can be found in many species,suchas all kinds of algae, higher plant, protozoan, prokaryote and archaea.We use thesequences among the species above to construct phylogenetic trees.The analysis ofthese trees suggests that，these genes originate from different ancestors,and theappearance of brown algae is a complex biological course.We have discussed the stability of8candidate reference genes in different tissuesand different growth periods of Saccharina japonica.We have obtained the Ctvalues,and determined their stability by statistical analysis of raw Ct values,and havegone further reseach by geNorm,NormFinder and Bestkeeper.Then,we have found indifferent tissues of Saccharina japonica,the ranking of the most stable gene to theleast stable gene by geNorm is: EF1α,UBA80,TUA,GAPDH,TUB,UBA52,18S, Actin；the ranking of the most stable gene to the least stable gene by NormFinder is: EF1α,UBA80,TUA,GAPDH,TUB,UBA52,18S,Act,and the most stable genes workedout by Bestkeeper are UBA80and EF1α.However, Actin is the worst candidatereference gene in this condition.Take all these results into account, EF1α and UBA80is the best group in different tissues.And in the different growth periods of SaccharinaJaponica, the ranking of the most stable gene to the least stable gene by geNorm is:UBA80,TUB,EF1α,UBA52,TUA,Actin,GAPDH,18S; the ranking of the most stablegene to the least stable gene by NormFinder is: UBA80,TUB,EF1α,UBA52,TUA,Act,GAPDH,18S, and the most stable genes worked out by Bestkeeper are UBA80andTUB.Although18S is very stable itself，it has the lowest comparison with othercandidate genes.So it isn’t the suitable reference gene in this reseach.Because18S isstable itself,we calibrate other genes with18S，then we will get the relative expressionof other genes.To calculate the average value，the SD value and the CV value,we willget a new ranking of the candidate genes expected18S.The result was that UBA80and UBA52are the most stable genes through this method.We also study the differenttissues and the different periods together by NormFinder,the result is that we can useUBA80and TUB together to calibrate the qPCR in both the different tissues and thedifferent periods.