Cloning And Functional Characterization Of The Fruit Aroma Related Candidate Genes From Mangshanyegan(Citrus Nobilis Lauriro)

Mangshanyegan (Citrusnobilis Lauriro) is a precious wild citrus germplasm resources, It had been already the characterized aroma of mangshanyegan fruit peel oil as β-myrcene and linalool oxides GC-MS analysis, with a notable content of linalool. Increasing with the living standard, consumer’s demand for the quantity and quality of citrus fruit also increased gradually. More and more people have been attracted by the fruit aroma research following with the application of GC-MS. But reports on the enzymes of metabolic regulation of aroma components and aroma formation and transformation mechanism are limited. Traditional identification of genes characterization has been hampered by the long time, complicated substrate supply in vivo. However, enzyme assays in vitro provided a beneficial and an effective method to identify genes involved in citrus aroma characterization.In this study, in order to reveal the molecular mechanism conferring high β-myrcene and linalool accumulation in the fruit of Mangshanyegan, myrcene synthase and linalool synthase genes cloned from the wild germplasm were investigated, protein obtained afterwards from the heterologous over-expression in Escherichia coli (E.coli), were subjected to in vitro functional characterization. The main results are as follows:1. Sequence analysis of the full length cDNA of MYRS and LIS revealed that there were one MYRS gene and two LIS genes (LIS-L and LIS-S) cloned from Mangshanyegan. In randomly collected20clones, the frequency of two LIS genes in Mangshangyegan was similar. There were different in37bp between two LIS genes, including a33bp insertion and4different nucleotides. MYRS and LIS genes have the common motif RRx8W and DDxxD for monoterpene synthases.2. qRT-PCR revealed that the MYRS were highly expressed in the mature Mangshanyegan fruit flavdo than in albedo, segment membrane and juice sacs, with as much as92.55,859.62and60.35times, respectively.3. The prokaryotic expression vector pET-CsLIS-L-fl, pET-CsLIS-L-tr, pET-CsLIS-S-fl, pET-CsLIS-S-tr, pET-CsMYRS-fl and pET-CsMYRS-tr were constructed and transformed in E.coli, respectively, the protein of full length (fl) and truncated (tr) version of MYRS and LIS were obtained subsequently.4. The His-tagged protein of truncated version (tr) and full length (fl) of MYRS and LIS were purified, respectively, and the system of enzyme assays in vitro was then constructed. Terpene products were then identified with GC-MS after GPP or FPP were added to the assay. It was found that the protein of truncated version of MYRS and LIS-S could catalytic GPP, but the protein of LIS-L had lost the function.5. Sequence analysis among the full length cDNAs of LIS in’Guoqing No.l’ Satsuma mandarin (C. unshiu Marc), Sweet orange (C. sinensis Osbeck) and ’Mangshanyegan’ revealed, that there were two LIS genes in Mangshanyegan while only one LIS in’Guoqing No.1’and sweet orange, respectively.In our study, we describe the molecular mechanism of the characterization aroma substance synthesis. The in vitro system established in the study provided an effective way to character the function of terpene synthase related genes. The optimization of in vitro method may assist further research on aroma synthesis.