The Selection Of Reference Gene And Expression Analysis Of Oil Content Enzyme Gene In Camellia Oleifera

Camellia oleifera is one of the high quality oil tree originated in China, it is called "Oriental olive oil" for the rich content of the unsaturated fatty acids in its seed oil. The unsaturated fatty acid content in good Camellia oleifera cultivars oil is up to90%(with oleic acid and linoleic acid, linolenic acid, also contains a small amount of high unsaturated fatty acids), far higher than the rapeseed oil, peanut oil and soybean oil. In the process of plant growth, gene regulation of lipid synthesis:SAD is the key gene synthesis oleic acid regulation in oil; GPAT, LPAAT, PAP, DGAT gene and PDAT is TAG synthetic genes, the DGAT is the speed limit enzyme of TAG synthesis in Kenendy pathway, and PDAT is the key gene TAG synthesis of another pathway; FAD mainly catalyzed polyunsaturated fatty acid synthesis, in which the FAD2and FAD6is the key gene regulation of oleic acid by catalyzing the formation of linoleic acid, FAD3and FAD7is the key gene regulation of linoleic acid by catalyzing the formation of linolenic acid in plants, but they have multiple copies.At present the camellia seed maturation cDNA library and EST library construction has been completed, and has obtained the expression abundance in mature seed fat synthesis related gene, on this basis, has completed the cloning of SAD, DGAT1, FAD2and FAD6. This research mainly completed:(1) Method for extracting total RNA from different organs/tissues of Camellia oleifera: Ambion(?) RNA extraction kit+CTAB, after agar gel electrophoresis and spectrophotometer, good quality RNA.(2) In Camellia oleifera transcriptome data basis of Camellia oleifera reference genes were screened from oil Camellia oleifera camellia seed kernel, can be applied to the analysis of the internal expression of candidate genes ALB, EF1α, EFβ, ETIF3H, UBC; can be used in Camellia root, stem, flower and seed of candidate reference genes expression of EF1α, can applied to the camellia root, stem, leaf expression of candidate reference gene ETIF3H analysis.(3) Based on Camellia oleifera EST data and lipid synthesis related cloned genes, through RT-qPCR, searching for the relation between the Camellia oleifera cultivars "Asus" fat synthesis with enzyme gene expression:I. Fat synthesis related enzyme genes in different growth period of Camellia oleifera seed is from high to low as follows: FAD2, PDAT, PAP, DGAT, FAD7, FAD6, SAD, FAD3, LPAAT, GPAT. The gene SAD, LPAAT, PAP, DGAT, PDAT and FAD in42weeks after blooming appeared peak expression, consistent variation of this change and oil content and oleic acid content, but the regulation mechanism of GPAT gene in lipid synthesis needs further study; at the same time, linoleic acid and linolenic acid content decreased, presumably because the synthesis rate of oleic acid in the oil is far greater than the rate of oleic acid into linoleic acid, and linolenic acid conversion rate will be affected, because the number of copies of the FAD gene, so it needs further research about FAD gene regulation mechanism.II. Analysis of lipid synthesis related gene expression and seed oil content and fatty acid composition of association analysis, by using the grey correlation method shows: grey relational grade oil content. A) Fat synthesis related gene and camellia seeds from high to low was: DGAT (0.9494), PDAT (0.8356), FAD7(0.7716), GPAT (0.5062), LPAAT (0.5038), PAP (0.5024), FAD2(0.5016), FAD3(0.5014), FAD6(0.5009), SAD (0.5004), the results proved that DGAT gene and PDAT gene in Camellia oleifera seed and oil composition has a close relationship, and found that the polyunsaturated fatty acid biosynthesis gene FAD7in lipid synthesis has also played a certain effect. For the grey relational grade oleic acid content. B) SAD gene and camellia seeds:0.7853, results are not consistent with the rules of the changes of the content of oleic acid synthesis, relationship between oleic acid in this study, SAD gene and Camellia oleifera seed still in need of further study. C. The gray correlation FAD2and FAD6and Camellia oleifera seed of linoleic acid content were0.6897and0.5002, and the grey correlation degree of linolenic acid FAD3and FAD7in camellia seeds were0.8032and0.5472, this also shows that FAD gene expression regulatory model needs to be further explored in camellia seeds.III. Fat synthesis related enzyme genes in different organs/expression characteristics of Camellia oleifera tissues showed tissue specificity. Among these genes expression of SAD gene and FAD2gene in leaves were significantly more than other organs/tissues, the two genes controlling the synthesis of unsaturated fatty acids, and the content of unsaturated fatty acid in plant leaves higher, the resistance is stronger.