Zhejiang Camellia Anthocyanin And Fatty Acid Synthesis-related Gene Cloning And Expression Analysis

Camellia chekiangoleosa is an important species of genus Camellia belonging to thefamily Theaceae. The economic value of C.chekiangoleosa is largely due to its beautifulornamental flower. The flower is red which generally blooms between February and March. Inaddition, C.chekiangoleosa is used to produce high-quality edible oil from its seeds. Its oil isrich in unsaturated fatty acid. The economic value of its oil is the same as oil palm, olive,coconut, and is commonly known as “eastern olive oil”. In this research, the key enzyme genesof anthocyanins and fatty acid biosynthetic pathway were cloned, and their expression patternswere investigated. The main results were showed as following:1、454GS FLX Titanium platform was used to generate an EST dataset. About46,279sequences were obtained, and24,593were annotated. Fifty-six functional groups including17,952sequences were classified according to their molecular function, biological process, andcellular component ontologies. A total of3844unigenes assigned to285metabolic pathwaywere identified against the KEGG database. Using Blast search against the AGRIS,1,740unigenes were found homologous to599Arabidopsis transcription factor genes.2、Based on the transcriptome dataset, ten anthocyanin biosynthesis pathway genes (PAL,C4H, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. Themultiple alignment and phylogenetic analysis demonstrated that each gene was a member ofcorresponding gene family. Relatively high expression levels of all the genes were observed inthe leaf except CHI, whose expression was abundant in the seeds. Hence, it was hypothesizedthat the anthocyanins were mainly synthesized in the leaves and transport to the flowers andseeds. The temporal expression analysis showed that CHS1, CHS2, and CHS3were highlyexpressed in the bud stage; F3H, CHI, PAL, and UFGT were maximally expressed in thesemi-open stage; and the expression of DFR and ANS gradually increased with flowerdevelopment. There are many similar or different patterns in various species, This could beattributed to some important differences between the types of anthocyanins produced by eachspecies and their regulatory mechanism.3、Thirteen complete-length cDNAs encoding BC, BCCP, ACAT, ACP, KASⅠ,KASⅡ,KASⅢ, KR, HD, ER, KCS, SAD and FAD2were isolated from seed by RACE. The sequenceanalysis validate their correctness. Almost all genes were highly expressed in the middle ofseed development (Ⅲ toⅤ) except FAD2. FAD2was maximally expressed in the second stage,its expression level in other stages is very low.Study of fatty acid accumulation pattern in seed development by gas chromatographyindicated that contents of fatty acids changed a lot. In the early stages, the total content oflinoleic acid and linolenic acid was higher than oleic acid, but they declined withthe subsequent development of the seed and the proportion of oleic acid increased gradually.The content of palmitic acid and stearic acid were gradual declined in seed development. SADand FAD2involved in the the formation of unsaturated fatty acids, wherein SAD catalyzed oleic acid formation and FAD2relating to the synthesis of linoleic acid or linolenic acid. Theirexpression quantities are roughly identical to the change trend of oleic acid linoleic acid andlinolenic acid. Although genes were highly expressed in the third to fifth stage, the relativecontent of saturated fatty acids was decreased gradually. This is due to SAD and FAD2convertsaturated fatty acids to unsaturated fatty acids.