Cytological And Transcriptomic Analysis Of Self-incompatibility In Dendrobium Officinale And Cloning Of Genes

Dendrobium officinale Kimura et Migo,an herb of the genus Dendrobium,is a precious traditional Chinese medicine,which has good edible and medicinal value,and has been included in Chinese Pharmacopoeia.Previous studies have shown that the selfing seed setting rate of the genus Dendrobium is extremely low,with selfing incompatibility,the types of plants in the natural environment performance increase genetic diversity,to promote the evolution of species,but in the agricultural production in order to improve the seed setting rate,mostly using artificial cross-pollination between varieties,different germplasm resources of intraspecific hybridization in iron production of the genus Dendrobium,quality and medicinal value there is a big gap.In order to explore the biological mechanism of Dendrobium officinale self-incompatibility,this paper used electron microscopy and transcriptome sequencing technology to study the cytological manifestations and potential mechanism related genes of Dendrobium officinale self-incompatibility.This study first of all types of iron genus Dendrobium strain group and cross-pollination,the flowers were collected from flowers after pollination and cross-pollination 0 h,2 h,4h,8 h pistil,observation of pollen tube germination in style,and extracted without pollination control group respectively,pollinate the flowers and 4 h after cross-pollination pistil RNA,carries on the transcriptome sequencing,and groups of data were differentially expressed genes（DEGs）analysis and the database function annotation,looking for some types and iron caulis dendrobii relevant candidate genes,types of looking for relevant candidate gene cloning and bioinformatics analysis,The results can provide a basis for further research on self-incompatibility mechanism of Dendrobium officinale,and provide ideas for protection and utilization of high-quality Dendrobium officinale germplasm resources.The main research results of this paper are as follows:1.With scanning electron microscopy were observed by paraffin section of the tin Dendrobium officinale stem from the flowers and cross-pollination,0 hour,2 hours,4 hours and 8 hours of pistil,observing the situation on the pistil stigma and style of germination pollen tube growth,pollination results show that the flowers can grow in the style of pollen tube in 2 hours,but most of the pollen tube stop growing in 4 hours.The pollen tube of cross-pollinated Dendrobium officinale could germinate on the stigma and grow rapidly in the style channel and into the ovary.From these results,it was preliminately speculated that the self-incompatibility type of Dendrobium officinale was gametophyte self-incompatibility type.2.Respectively by the iron without pollination of caulis dendrobii pistil,the pistil of the flowers 4 hours after pollination,4 hours after cross-pollination the pistil of the transcriptome sequencing,obtain the original data contains 63,409,511,100base Sum,after removal of low quality sequence,get 211,365,037 read Sum,Q20,average Q30 and GC content are 97.56%,93.24%and 47.63%respectively,71.58%of the clean reads can be compared to a reference genome,Genes were sequenced using Swiss-Prot,NR,PFAM,KOG,GO,KEGG and COG database annotations,and38,373 functional genes were found to be annotated in these databases.1944,1758 and475 differentially expressed genes were screened between outcrossing group and control group,self-crossing group and self-crossing group,respectively.Again to GO annotation analysis of differentially expressed genes,in different group compared the control group,different selfing group compared the control group,and group contrast between selfing groups,there were 815,813,216 different genes annotated in the database,the key to GO between different alternating group contrast selfing,COG,rich concentrated KEGG function classification,some types may be related to the pathway and classified into database,the enrichment of including cationic transport,metal ions,RNA degradation,signal transduction mechanism,fatty acid biosynthesis and transcription pathway,On different alternating group contrast selfing differentially expressed genes between functional annotation analysis,screening the 41 types may have about the differences of genes,including six calcium signal gene（4 calcium binding protein gene Do CML,a calcium dependent protein kinase gene Do CPK,a similar protein calcineurin b gene Do CBL）,a specific protein rich in leucine repeated extension of pollen similar protein（Do LRX）,L lectin receptor kinase（Do L-RLKs）structure domain,Four ARC（armed repeat containing）similar genes（Do ARC）,eleven S site receptor kinase-like genes（Do SRK）,two extracellular vesicle component family genes（Do EXO）,nine ubiquitination related genes（Do EBI）,one fatty acid metabolism related gene（Do FAT）and six amino acid metabolism related genes（Do AMI）were identified.The transcriptome was also verified by q RT-PCR,and it was found that the differentially expressed genes and the transcriptome regulation trend were generally consistent with the multiple,indicating the reliability of transcriptome data.3.To transcriptome screening out of a possible types and Do EXO70B1 related gene cloning and bioinformatics analysis,found that Do EXO70B1 gene ORF length of 2148 bp,translation out of 727 amino acid sequence,the translation of protein molecular formula for C₃₅₇₉H₅₆₃₀N₉₉₄O₁₁₄₆S₂₆,molecular weight of 81.75 k Da,theoretical isoelectric point is 5.35,instability index of 48.74（>40）,protein secondary structure prediction,found that 57.50%isα-helix structure,37.69%is not curly structure rules,3.16%are elongated chain structures and 1.65%areβ-folded structures.By predicting the tertiary structure of the protein,we found that the tertiary structure of Do EXO70B1 is similar to that of Exo70A1 in Arabidopsis thaliana,suggesting that Do EXO70B1 plays an important role in self-incompatibility of Dendrobium officinale.In nucleic acid Blast and phylogenetic tree,we found that Do EXO70B1 was the most closely related to Phalaenopsis,closer to Brassica napus.These results could provide a basis for further expression studies and elucidate the role of Do EXO70B1 in the self-incompatibility mechanism of Dendrobium officinale.