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Transcriptomic Analysis Of Targeted Silencing Of ACC Oxidase Genes In Transgenic Banana

Posted on:2018-03-19Degree:MasterType:Thesis
Country:ChinaCandidate:Y XiaFull Text:PDF
GTID:2393330515495221Subject:Pomology
Abstract/Summary:PDF Full Text Request
Background:Among 18 ACC Oxidase homologous genes existing in the banana genome there are two genes,Mh-ACO1 and Mh-ACO2,which participate banana fruit ripening.To better understand the physiological functions of Mh-ACO1 and Mh-ACO2 involved in regulating the ripening process of banana fruits two paralogous genes in banana fruit ripening,highly homologous ACC oxidase genes,Mh-ACO1 and Mh-ACO2,have been seperately targeted for gene silencing by using RNA interference(RNAi)technology,two hairpin-type siRNA expression vectors targeting each of the Mh-ACO1 and Mh-ACO2 were constructed and transformed into banana genome by Agrobacterium-mediated transformation.The generation of Mh-ACO1 and Mh-ACO2 RNAi transgenic banana plants were confirmed by Southern analysis.To gain insights into the functional diversity and complexity between Mh-ACO1 and Mh-ACO2,transcriptome sequencing of banana fruits using Illumina next-generation sequencer was performed.Result:To get an overview of transcriptom characteristics of transgenic banana plant after the application of RNAi for the inhibition of ACC oxidase genes,a high-throughput RNA sequencing based on next generation sequencing(NGS)technology(Illumina)were performed.A total of 32,093,976 reads,assembled into 88031 Unigenes for 123617 transcripts were obtained.Total number of reads that can be mapped to the reference genome for the untransformed plant(WT),Mh-ACO1 silenced(As1),and Mh-ACO2 silenced(As2)transgenic plants are 78.50%,79.45%,and 79.99%,respectively.88031 Unigenes were generated and assigned to know protein databases including NCBI non-redundant protein database(nr)(42325,42.8%),Gene Ontology(GO)(24411,27.73%),NCBI nucleotide sequences(nt)(42325,48.07%),Swiss-Prot(24931,28.32%),euKaryotic Orthologous Groups(KOG)(12031,13.66%),Kyoto Encyclopedia of Genes and Genome(KEGG)(12163,13.81%)and Protein family(PFAM)(24104,27.38%).A total of 88031 Unigene 6300 Unigenes were successfully annotated in all the seven databases and 46904 unngenes were successfully annotated in at least one databaseits.as determined by Blastx search.Differential expression analysis of Asl-vs-WT(3050)As2-vs-WT(2784)and Asl-vs-As2(943)were compered using the DEGseq.Then the differentially expressed genes(DEGs)were analyzed by GO enrichment and KEGG enrichment.Gene Ontology(GO)enrichment analysis was performed to classify the gene function of Unigenes.Significantly enriched GO terms and the number of differentially expressed genes(DEGs)with GO annotation were 'Catalytic activity'(1327,56.4%),'Heme binding'(65,2.76%),'Tetrapyrrole binding'(66,2.81%),and'Oxidoreductase activity'(287,12.21%).According to KEGG annotation,most of the DEGs were enriched in 'Carbon fixation in photosynthetic organisms'(30,22.73%),'Cysteine and methionine metabolism'(32,21.19%),‘Citrate cycle(TCA cycle)'(25,22.73%),and 'Starch and sucrose metabolism'(49,16.67%).To gain a better understanding of the postharvest ripening of banana fruits at the molecular level,the genes related to ethylene biosynthesis and signal transduction were further selected for more comprehensive studies,also,the carbohydrate metabolism pathway of banana fruit during third maturation was analyzed.The majority of contig ID's shown by high throughput sequencing were similar at the stage 3 of fruit rIPening in banana where the transcriptome analysis in this research apply to.In order to validate the most significant expression profiles of the ethylene biosynthesis and signal pathway genes in Mh-ACO1 and Mh-ACO2 RNAi transgenic banana fruits at stages 1,3,5 and 7,quantitative Real-T ime-PCR was performed with mRNAs from both peel and pulp of banana fruits.The results showed that the direction of expression change of all selected genes based on qRT-PCRs agreed with those detected by high throughput sequencing.Both peel and pulp tissues showed the expression levels of a significant number of genes to be similar among banana samples of WT,As 1 and As2.These results suggest that both ethylene biosynthesis and signaling in ripening banana fruits may be controlled both in the peel and pulp tissues.Expression levels of genes related to ethylene signalling in ripening banana fruits were strongly influenced by the expression of genes associated with ethylene biosynthesis.Conclusion:the strategy applied in this research provides a useful tool for understanding the role and the networks underlying these genes in the process of fruit ripening.The transcriptomic analysis in this research also contributes to the banana funtional genomics studies in future.The transcriptome of the three samples and the DGEs database provide invaluable new date for study in banana fruit repening and carbohydrate metabolic pathway,and will lay the foundation for the banana functional gene analysis and research in future.
Keywords/Search Tags:fruit ripening, ethylene production, genetic transformation, RNA interferen
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