Transcriptomic Analysis Of The Protocorms In Dendrobium Officinale Kimura Et Migo

Dendrobium officinale Kimura et Migo,a kind of valuable traditional Chinese medicine,is a perennial epiphytic herb of Dendrobium Orchidaceae.However,because of the special protocormdevelopmentof D.officinale,the seeds germination rate were very low in natural conditions.The protocorm development whose maturation sends contained the immature embryos need more nutrition to germinate and it maked become endangerous.In this paper,D.officinale was used as the material to study this four aspects which contained the morphological process of the protocorm,the sequence analysis of transcriptome,the analysis of differential expression gene of transcriptome and the DoTrx L2 sequence analysis of expression spectrum.The main experimental results are as follows:(1)The morphological which changed in the development of protocorm were observed by paraffin section and electron microscope to identify the morphological characteristics of various stages during the process of protocorm development.We found that it is necessary to break through the two layers which were the inner and outer testa in the process of the proembryo activationprocess.The cells in the stage of the protocorm formation were polar and there were villiform cells appeared in bottom of protocorm.The stomata and dorsum of protocorm appeared during the formation of the primary meristem.And a line of cells occuredbetween thepromeristem and apical meristem.(2)In this paper,Illumina sequencing technology was used to sequence the transcriptome different developmental stages and different tissues of D.officinale.The different development stages were seed embryo activation and breakthrough testa,protocorm,promeristem,SAM,spheroidicity protocorm,leaf primordium and vascular system and RAM.The different tissues contained protocorm-like bodies,callus and seedings.There were 15 samples were sequenced by Illumina RNA-seq and 106.81 Gb Clean Data was obtained whose Q30 reached 91.20%.The sample F01 was sequenced by the three generation transcriptome and obtained 13.86 Gb clean data.There were 381,755 ROI(reads of insert)sequnces and 194,404 FLNC sequences were obtained.Using the RS_IsoSeq module of SMRT Analysis to clustered and analyzed the full length sequence,79,477 consistent transcriptional sequences were obtained and 60,726 high quality full length transcriptional sequences were acquired by sequences correction which did not have full length.These unigene sequences were analysised,including structure and fuction annotation.The structure analysis contained alternative splicing,SSR and SNP.And the fuction annotation contained GO analysis,COG analysis,KEGG analysis and so on.(3)We sequenced the protocorms of D.officinale at different developmental stages and obtained transcriptome database.21,492 differentially expressed genes were screened according to the data of the transcriptome.This unigene sequences were compared and annotated with the common database of protein,including the Nr,GO,COG and KEGG,which showed that the most gene annotation was metabolic activity.Indicating these genes made metabolism active during the development of protocorm and regulate the growth and development of protocorm of D.officinale.Base on these unigene sequences,78 sequences of TRX were screened out and 11 genes were differentially expressed genes.These 11 differentially expressed genes were analysised which were in different developmental stages of protocorm of D.officinale.(4)The Thioredoxin-like 2 gene(DoTrxL2)in D.officinale was cloned by RACE and nest PCR technologies.The full length of DoTrxL2 contained an open reading frame(ORF)of 639 bp which encoded 213 amino acid residues,whose product contains a conserved CXXC domain.Phylogenetic analysis showed that the DoTrxL2 has the closest relationship with monocotyledons,such as Phalaenopsis equestris and Asparagus officinalis.qRT-PCR analysis showed that the expression levels of DoTrxL2 were significantly varied in different developmental stages of protocorm and different tissues,which indicated that the DoTrxL2 plays an important role in the development of protocorm and the morphogical establishment of tissues in plant.