The Family Analysis Of Ubiquitin Activator E1and Ubiquitin Conjugating Enzyme E2 Family And The Cloning,Expression Analysis Of DoUBC9 In Dendrobium Officinale Kimura Et Migo

Dendrobium officinale Kimura et Migo is a perennial herb monocotyledonous plant of the genus Orchidaceae,which has high medicinal and ornamental value.officinale has a common bulb development pattern shared by orchids,that is,when the seeds mature,their embryos remain in the undifferentiated stage,and they need to undergo protocorm period to develop into a complete plant.Due to high demands on the natural environment and the artificial cockroach picking,now it has become an endangered species.This study was divided into four aspects,the morphological observation of officinale in different periods,the identification and analysis of the ubiquitin-activating enzyme E1(DoUBA)family in officinale,and the identification of the ubiquitin-conjugating enzyme E2(DoUBC)family in officinale,and the identification and analysis of the cloning of DoUBC9.The main experimental results are as follows:(1)The development of protocorms generally go through seven periods(P1~P7),which are:breakthrough of seed coat after embryo activation,formation of protocorms,original meristems,apical meristems,ellipsoidal protocorms,vasculatures of leaf primordia and root-end meristems.The protocorms of each period had to be selected,and the morphological observation was recorded using stereoscope and scanning electron microscope.(2)Ubiquitin-activating enzyme E1 is the first enzyme involved in the ubiquitin-proteasome pathway.It is responsible for the activation of target proteins,and plays a key role in the ubiquitination pathway.This study used the 3~rdd generation transcriptome to measure sequence,and obtained four DoUBA family members from the sequence files annotated by GO analysis,KEGG analysis,etc.All sequences go to the redundancy by using SMART and CDD domain queries.By analysising the predicted protein,all of them are fat-soluble hydrophobins,except DoUBA2,all of them are acidic proteins,and they are all stable proteins except DoUBA3.The subcellular localization has a large probability distribution in lysosomal,nucleus and chloroplasts,and there is no transmembrane domain.The secondary structure has the largest proportion of?-helix and random coil.FPKM was used to quantitatively analyze each gene.DoUBA1,DoUBA2 and DoUBA3 were highly expressed in various periods,while DoUBA4 was extremely low in P2 and P4.(3)The ubiquitin-conjugating enzyme E2 is the second enzyme involved in the ubiquitin-proteasome pathway and is responsible for the transmission of target proteins after activation.It belongs to intermediate enzymes and has a large number of gene families.In this study,62 ubiquitin-conjugating enzyme E2 family members were identified by constructing a local database of officinale genome using Blast,local alignment and HMMER methods in combination with SMART and CDD domain validation.In analysis of gene structure,the 62 family members contain 0-15introns;in protein motif analysis,most of the proteins contain one or two motifs in motif1,motifi2,and motifi3;in the phylogenetic analysis,62 DoUBCs and UBC of Arabidopsis,maize and rice is divided into 15 subfamilies.Each subfamily's DoUBC gene has a relatively conservative motif.The protein sequences ware completely aligned,not only found the UBC family common"HPN"and"PxxPP"conservative protein sequence,but also the"D/NP"conserved sequence unique to officinale was found;Through the analysis of protein biochemical characteristics,62 proteins are non-lipid soluble hydrophilic proteins,and all acidic proteins,most of them are stable;Using the qRT-PCR to analyze the expression of the DoUBC family,except 7 genes were not expressed or expressed at the P1~P7 protocorm stages,all the remaining 55genes were widely expressed in the protocorms at various stages.The expression levels of 23 genes decreased first and then increased and the expression levels of 22genes increased first and then decreased.Most of the genes had the lowest expression at P5.In addition,most of the genes expressed the most in P3 and P7.(4)In order to study the molecular mechanism of ubiquitin-conjugating enzyme UBC9 in the development of protocorm of officinale,this study used bioinformatics to identify the UBC9 gene from the obtained officinale genome,named DoUBC9.RACE was used to clone the 3'end of DoUBC9 and the complete sequence was obtained by splicing with the genomic sequence.The CDS of DoUBC9 is 447 bp in length,encoding a total of 148 amino acids with 3 exons,and the exon plus intron is7,176 bp.The protein encoded by DoUBC9 is an acidic,unstable protein with a molecular weight of 16,436.97 Da,and the isoelectric point(pI)is 7.76,in vitro half-life is 30h;in the secondary structure,?helix,extended chain,random coil,?-sheet each accounted for 34.46%,18.92%,39.19%,7.43%;subcellular localization prediction results show that DoUBC9 large probability distribution on the secretory vesicles and plasma membrane.Sequence alignment and phylogenetic tree analysis indicated that UBC9 from officinale and other species had highly conserved UBCc domains,and the evolutionary relationship was highly conserved.qRT-PCR analysis showed that DoUBC9 had the highest expression in callus and the lowest expression in leaves in different tissues.During protocorm development,it had the highest expression in P1 and less expression in other periods,indicating that the gene may promote rapid cell division and growth of plants.