Identification Of Genes Related To Oil Gland Development And Essential Oil Biosynthesis In Kumquat

Kumquat?Fortunella spp.?is an evergreen fruit plant of genus Fortunella,subfamily of Aurantioideae,which originated in China.Fortunella is the closest genus of Citrus.Citrus essential oil is composed of a variety of components existing in oil gland of peels and other tissues.It is the major source of plant essential oils.Kumquat fruit is the main citrus fruit,which the peel can be eaten together with pulp.The essential oil in peels have a significant impact on taste,flavor and human health.Up to now,the genetic regulation on oil gland formation and essential oil biosynthesis in kumquat were rarely studied.In this thesis,a kumquat mutant with few oil gland was used to study the genetic factors related to oil gland formation and development,essential oil biosynthesis and accumulation.The main results were as follows:?1?Identification of genes related to oil gland development in kumquatAccording to the phenotypic traits,the female parent Luowen kumquat,male parent Huapi kumquat and their hybrid F₁ population were analyzed.The extreme phenotypic traits population?high density pool and low density pool of oil gland?were constructed for re-sequencing combined with BSA analysis,and RNA-sequencing to identify the genes related to oil gland development.Ten genes were selected by Re-sequencing combined with BSA analysis.Through the homology analysis,carboxyesterase gene Ciclev10005338m.g and Cysteine proteinases superfamily protein gene Ciclev10005334m.g were involved in plant programmed cell death.Phosphorus induced protein gene Ciclev10005370m.g and hydroxyl methylglutaryl?HMG?-CoA synthase gene Ciclev10005099m.g were involved in secretory cell division at early phase of oil gland development.pectin methylesterase inhibitor superfamily gene Ciclev10004719m.g and MYB85 protein gene Ciclev10005666m.g may be involving in the sheath cell wall formation of mature oil gland.Four genes,cytochromeP450geneCiclev10004733m.gandCiclev10006782m.g,Glucose-methanol-choline?GMC?oxidoreductase family protein gene Ciclev10006541m.g,Peroxidase gene Ciclev10005804m.g,were involved in plant growth and development,but the specific metabolic pathway of these genes have not been reported.Four genes were chosen as candidate genes by differential expression genes analysis of RNA-sequencing.Through the homology analysis,HXXXD-type acyltransferase genes,Ciclev10011796m.g,Ciclev10024067m.g and Ciclev10028504m.g,may be affect the oil gland development and essential oil accumulation.Glucosidase gene Ciclev10031212m.g may be intermediating the release of terpenoids which exist in the form of glycosides to affect the same metabolic process mentioned in previous sentence.?2?Cloning and diversity analysis of kumquat d-limonene synthase gene?FcLS?Limonene is the main component of citrus essential oil.Based on the homologous cloning combine with RACE technology,the cDNA sequences of 18 d-limonene synthase?LS?genes with high similarity were isolated from Rongan kumquat?GenBank accession number:MH537603-MH537620?.Sequence analysis demonstrated the full-length ORF of FcLS were 1821bp.They were encoded the hydrophilic protein with 606 amino acids,and they had a N-terminal chloroplast signal peptide with 25 amino acids.Conserved motif DDxxD,NST/DTE and RR?x?8W were all existed in citrus LS-like genes by homology analysis.FcLS were highly expressed in kumquat peel,which showed the obvious tissue specificity of FcLS.Using the re-sequencing and DGE profile data to blast these homology sequences,it showed that the variation of FcLS were derived from the change of 12 SNPs.The synonymous mutant in some SNPs of FcLS resulted in the production of 12 FcLS.And the re-sequencing data also showed more information of SNPs than RNA-sequencing.In addition,the three-dimensional structure prediction showed SNPs could not change the crystal structure of FcLS,indicating FcLS were highly conserved in kumquat.In a words,kumquat was not only significant sequence difference between FcLS and citrus LS-like gene,but had multicopy duplicate genes with SNP differences.?3?Functional characterization of kumquat d-limonene synthase gene?FcLS1₁?The prokaryotic expression vector pET28a-LS with His-tag was constructed using the sequence of FcLS1₁,and transformed into E.coli Transetta?DE3?to express the recombinant protein.Then the recombinant protein FcLS1 was isolation and purification by nickel column affinity chromatography method.Based on the related reaction of terpenoids synthesis pathways,the recombinant protein FcLS1 were used for enzymatic reaction with substrates GPP,FPP and GGPP.Then GC-MS was used to identify the function of the volatile enzyme products.It turned out that FcLS1 was an active soluble protein.It reacted specifically with GPP to produce large amounts of d-limonene.That is,FcLS1₁ was a gene encoding an active d-limonene synthase.