Selection Of Reference Genes For Gene Expression Studies In Tartary Buckwheat Using Quantitative Real-time PCR

Fagopyrum tataricum,also known as tartary buckwheat,is in the genus Fagopyrum in the family Polygonaceae.It is a kind of small grains used as medicine and food because of its rich flavonoids with the represent of rutin.Meanwhile,due to its characteristics of growing environment,tartary buckwheat has a strong tolerance to a variety of adverse conditions,such as drought,UV-B radiation and cold.Therefore,it is a hot field to study the molecular mechanism of stress resistance and flavonoid metabolism in tartary buckwheat.At present,Ft Actin（Actin 2 genes）,Ft SAND（SAND protein family）and Ft H3（histone 3）gene were be used as the reference genes for gene expression analysis in tartary buckwheat.If only the above three genes are used as the internal reference genes,which may lead to an inaccurate or even opposite results for target gene expression analysis in tartary buckwheat.Therefore,it is very important to screen and identify the stable reference genes under various experimental conditions,which is help to provide the more accurate and suitable internal reference genes for gene expression level analysis for tartary buckwheat.In this study,based on the tartary buckwheat’s transcriptome data and the traditional housekeeping genes,Ft SAND,Ft CACS（clathrin adaptor complex subunit protein family）,Ft Expressed1（unknown protein family）,Ft Actin,Ft H3,Ft EF-1α（elongation factorα）and Ft GAPDH（glyceraldehyde-3-phosphate dehydrogenase）were chosen to be the candidate reference genes in“Xi Qiao No.2”,a local variety of tartary buckwheat.After the expression levels of the above genes in tartary buckwheat with the different experimental conditions were analyzed by real-time quantitative PCR technology,their expression stability were further analyzed using three softwares Ge Norm,Norm Finder and Best Keeper.The main results are as follows:1.According to the transcriptome data,the c DNA sequences of Ft Actin,Ft GAPDH and Ft EF-1αgenes were cloned from tartary buckwheat.Sequence analysis showed that Ft Actin,Ft GAPDH and Ft EF-1αhad an open reading frame with 1134,1034 and 1347 bp,respectively.The further analysis indicated that nucleotide sequences of Ft Actin showed 80%homology with Cryptomeria japonica,Ft GAPDH showed 80%homology with Fagopyrum esculentum,and Ft EF-1αshowed 80%homology with Camellia oleifera.2.The cotyledons and hypocotyls were sampled from the seedling tartary buckwheat after treatment with drought,cold,UV-B and saline,while roots was gathered after aluminium treatment.Moreover,roots,stems,leaves,flowers and immature seed were collected at the immature seed stage,respectively.The expression levels of seven candidate reference genes,Ft SAND,Ft CACS,Ft Expressed1,Ft Actin,Ft H3,Ft EF-1 and Ft GAPDH,were measured by RT-q PCR.And then,the stable reference genes were selected according to the evaluation of the stability analysis software.Firstly,the Ct value comparison implied that the highest average expression was Ft EF-1α（Ct=17.61）,and the lowest was Ft SAND（Ct=25.6）.Because the greatest Ct value variability was found in Ft Actin（SD=1.83）and Ft GAPDH（SD=1.49）,it meant they were the unstable internal reference likely.However,Ft H3 could be taken as the most stable internal reference gene due to the least Ct value variability（SD=0.79）.Then,the stability analysis software Ge Norm,Norm Finder and Best Keeper analysis showed that Ft H₃ and Ft CACS in cotyledons and hypocotyls,and Ft SAND、Ft H₃、Ft Expressed1 and Ft CACS in the mix samples of hypocotyls and cotyledons were the most stably expressed genes under drought treatment.Ft H₃ and Ft CACS were the most stably expressed genes under cold conditions.Ft H₃,Ft Actin and Ft Expressed1 in cotyledons,Ft SAND,Ft H₃and Ft CACS in hypocotyls,and Ft CACS in the mix samples of hypocotyls and cotyledons were the most stably expressed genes under UV-B treatment.Ft H₃and Ft CACS in cotyledons,Ft H₃,Ft CACS,Ft Expressed1,Ft H₃and Ft SAND in hypocotyls,and Ft Expressed1 and Ft SAND in the mix samples of hypocotyls and cotyledons were the most stably expressed genes under Na Cl treatment.Seen from the all the treatments,Ft CACS and Ft SAND in cotyledons,Ft SAND in hypocotyls,and Ft SAND in the mix samples of hypocotyls and cotyledons were the best reference genes.Moreover,Ft Expressed1 and Ft EF-1αare the best reference genes for aluminium treatment.In addition,Ft CACS and Ft Expressed1 could be taken as the suitable reference genes at the immature seed stage of tartary buckwheat.In summary,the stably expressed genes could be used as the internal reference genes for tartary buckwheat under the corresponding experimental conditions.3.Both stable and unstable reference genes mentioned above were used as the internal control to analysis the relative expression of two genes,in which Ft STAR is an Aluminum responsive gene,and Ft DFR（dihydroflavonol 4-reductase）encodes a key enzyme involved in flavonoid metabolism of tartary buckwheat.The expression analysis in roots showed that Ft STAR was expressed increasedly under aluminum stress,but the relative expression level was influenced by reference gene.If the stable expression gene Ft Expressed1 was used as reference gene,the Ft STAR expression level was increased of approximately 6.24 fold after aluminum treatment.When the unstable gene Ft Actin was used as reference gene,the Ft STAR expression level was only increased of approximately 2.48 fold.Furthermore,if the stable reference of Ft CACS,Ft H3 and Ft Actin were taken as internal control under UV-B treatment,the Ft DFR expression in hypocotyls showed the same trend of change as the above reference genes,i.e.the expression level was gradually decreased with the stress time.When the unstable gene Ft EF-1αwas taken as reference gene,the Ft DFR expression level showed an irregular fluctuation.