Selection Of Internal Reference Genes In Protocorm And The Expression Analysis Of LEA Gene Family Of Dendrobium Officinale Kimura Et Migo

Dendrobium officinale Kimura et Migois is perennial herb,belongs to Dendrobium,Orchidaceae and the embryogenesis of it was at a standstill at globular stage when the seed matured and body plan is constructed through protocorm development.The construction of protocorm included embryo activation?EA?,protocorm formation,protocorm maturation and protocorm degeneration.The stages of protocorm development could be divided into 8 stages:seed embryo activation and breakthrough testa;protocorm;promeristem;SAM;spheroidicity protocorm;leaf primordium and vascular system;RAM;degeneration and root/leaf.The analysis of the nucleotide across all protocorm stages showed that the content of nucleotide rised slowly at the former 5 stages and rised greatly after spheroidicity protocorm.The using of internal reference genes could significantly influence the expression level of specific genes.In this study,the 26 most stably expressed genes in whole transcriptome of protocorms'and 5 housekeeping genes from literature were used as tested genes.Then 19 tested gene were selected as candidate reference genes from the primary express stability analysis of all 31 tested genes and screened with 4 application softwares?geNorm,NormFinder,BestKeeper and RefFinder?.The results showed that the novel reference genes from transoriptome were stabler than the tranditional housekeeping genes across all samples.The determination of optimal reference genes showed that a few reference genes could effectively normalize expression level of specific genes in protocorm development and the optimal top 2 reference genes were ASS and APHlL.Meanwhile,validation of GNOM,AP2 and temperature induced gene?TIL?for normalization demonstrates the usefulness of the validated candidate reference genes.The expression profiles of these genes varied under protocorms and temperature stress according to the stablest and unstablest reference genes,which proved the importance of the choice of appropriate reference genes.LEA was late embryogenesis abundant protein involved inthe embryogenesis formation of plant and resistance to stress and other physiological process.This study built a local database for blasting with the genome of D.officinale and then scaned the whole genome by blast+ and HMMER.58 certain LEA genes were achieved after the identification of its typical domains by CDD and SMART databases,which included 7 domains:LEA₁,LEA₂,LEA₃,LEA₄,LEA₅,Dehydrin and SMP and could be divided into 7 subfamilies according to their domains.The structures of LEA genes showed that 37 LEAs had no intron and the intron numbers of others betweens 1-4.The analysis of motif region indicated that the members of the same subfamily had relatively consistent motif characteric.The majority of LEAs could be expressed and they were different at different development stages of protocorm.Moreover,their expression level had significant rising after the stage of seed embryo activation and breakthrough testa?P1?and 15 LEAs were predominantly expressed at,the stage of promeristem,in which,DoLEA45 and DoLEA52 had the top expression level.The up-regulated expression genes were about half in low temperature and PEG stress,however,almost all LEAs showed up-regulation tendency in ABA stress.From the analysis above,LEAs may play an important role in the morphogenesis of protocorm of D.officinale and resistance to low temperature and drought and so on extreme conditions.