Selection Of Reference Genes For Gene Expression In Rhododendron Delavayi Franch

Rhododendron delavayi Franch.is a perennial alpine woody flower of Rhododendron Subge.Hymenanthes,it has a high ornamental and medicinal value.As an important parent of original breeding,it has an important value for scientific research,development and utilization.So far,the whole genome sequence of rhododendron delavayi has been published,which is bound to promote the rapid development of rhododendron in the field of molecular biology research.However,there are few studys on the selection of gene expression reference genes.In this study,we selected Rhododendron delavayi as material,and selected 11 candidate internal reference genes(GAPDH,Actin,EFla,Tubulin a-5,Ubiquitin,UEP,UECl,UEC2,TIP41-LIKE FAMILY PROTEIN,Tubulin beta,,TATAbinding protein)in woody plants,used Quantitative Real-time PCR to analyze the expression stability of these 11 candidate internal reference genes in different developmental stages of adult and young plants of Rhododendron delavayi and in drought stress.Based on the analysis results of qBASE and NormFinder,the best internal reference genes or combinations were selected for different test sample sets.The research results are as follows:1.In this study,we selected the 11 common candidate internal reference genes,the detection results of Quantitative Real-time PCR show that the corresponding target fragments could be amplified in the genome of Rhododendron delavayi,the amplification specificity and amplification efficiency(94%-106%)met the primary conditions of internal reference gene screening.2.In this study,we comprehensively analyzed the expression stability of 11 candidate internal reference genes in adult plants(mature leaves,flower buds,pistils,stamens and petals)and young plants by qBASE and NormFinder internal reference gene stability evaluation software.In the set of mature leaf samples,the optimum combination of internal reference genes was GAPDH+ Actin.In the set of flower bud samples,the optimum combination of internal reference genes was Tubulin-β + UEC1.In the set of different flower tissue and organ samples,the optimum combination of internal reference genes was EF1α a Tubulin-β.In the set of different tissue parts of Rhododendron delavayi adult plant,the optimum combination of internal reference genes was UEC1 + GAPDH+ TIP41 + UEP.In the set of different tissue samples of young plant,the optimum combination of internal reference genes was TATA + UEC1 ＋Actin.In the set of different tissue samples of adult and young plants,the optimum internal reference gene combination was GAPDH+ UEC1.3.We selected the internal reference gene of GAPDH which has a good stability to detect the expression of drought stress response gene NCED1 under different drought stress treatment times.It was found that the expression trend of NCED1 was in line with the expectation during the period from day 9 to day 13.On this basis,the optimum internal reference gene combination was Actin + UECI,the combination of internal reference genes with poor expression stability is TIP41 + UEC2,by comparing the expression combination of the two internal reference combination in samples treated with different drought stress times,the expression patterns of drought response gene NCED1.The results showed that whetherActin or UEC1 were used as a combination or a single internal reference,the expression abundance of NCED1 was higher,and in line with the expected expression trend.The above results promote the research progress of internal reference genes in Rhododendron delavayi,and provide some technical references for the research on the selection of internal reference genes for gene expression of other Rhododendrons.