Analysis Of Gene Expression On Ethylene Biosynthesis Of Akebia Trifoliate(Thunb.)Koidz In Ripping Process

Akebia trifoliate(Thumb.) Koidz called as ‘bayuegua', ‘bayuezha', ‘bayuecha' or‘niuyaozi', is a woody liana, belonging to Akebia Decne of Lardizabalaceae. Although the fruit of A. trifoliate is not widely eaten due to its thick pericarp, dehiscence and full of seeds, it can be used as a traditional Chinese medicine. Ethylene plays a regulative role in the Ripening process accelerated by the accumulation of ethylene,which is generated by the cycle of Methionine. The key enzyme of ethylene synthesis are methionine adenosyl transferase, ACC sythase and ACC oxidase. To unveil the regulative accumulation of ethylene during the ripening process, the main content of this research includes:1. To explore gene expression of A. trifoliate in ripening process, six libraries from different fruits statges of A. Trifoliate were sequenced by Illumina Hi Seq2000. After trimming the adapters and removing contamination, the transcriptome of A. trifoliate is de novo assembled using Trinity. The experiments acquired 106,547 unigenes in total among which 39,501 unigenes hit the sequence in at least one database, i.e.27,302 and 12,014 unigenes found their homologs in Gene Ontology(GO) and eu Karyotic Orthologous Groups(KOG), respectively. Based on the annotation in Kyoto Encyclopedia of Genes and Genomes(KEGG), 11749 unigenes were annotated and predicted to involve in 270 KEGG pathways.2. Results showed that, the cross section diameter of fruits increased rapidly within the 42 days once it bears fruit. In addition, the longitudinal diameter increased rapidly within the 35 days once it bears fruit. According results above, fruit of A. trifoliate grew rapidly within the 42 days once it appeared. The Sample 1 could be classified into the fertilization, the Sample 2 could be classified into the rapid-growing, the Sample 3 could be classified into the slow-growing, the Sample 4 and Sample 5 could be classified into the fulling, the Sample 6 could be classified into the premature growing.3. To figure out the most stably expressed genes in fruit ripening process, 22 candidate reference genes from transcriptome of A. trifoliate were selected for stability analysis using ge Norm, Normfinder and bestkeeper. According to results,using histone h3 with a combination of EF-? as reference gene could accurately indicate relative expression of other genes. As a result, suitable reference genes combination of A. trifoliate in fruit ripening process was first investigated which increased the accuracy of gene quantification.4. To validate the sequence accuracy of unigenes, 13 target genes involved in ethylene biosynthesis were cloned and sequenced. High similarity was observed for them.Expression of these genes was also validated using RT-q PCR. No significant differetial was observed between RT-q PCR and transcriptome data.5. Based on transcriptome data, expression pattern of 13 genes from above were investigated. The results showed that, during the fruit ripping, m RNA levels of methionine adenosyl transferase and ACC synthase were decreased, and the expression of ACC oxidase gene was significantly upregulated in Sample 4.Measuring by HPLC, ACC oxidase gene was suggested playing the most important role in the accumulation of ACC.