| Fatty acid biosynthesis is an important metabolic pathway in cell metabolism.The fatty acid is an important component of cell membrane lipids.The level of unsaturated fatty acids affects the fluidity and stability of cell membranes,and it is closely related to plants resisting stress.At the same time,fatty acid is also an important component of oil crop seed oils.Tea seed oil has a high ratio of oleic acid and linoleic acid and is beneficial to human health.It is a high-quality vegetable oil.In this study,tea leaves were used as materials,RT-PCR and RACE technology were combined to clone different key enzyme genes in the anabolic pathway of saturated fatty acids in tea tree and the key rate-limiting enzymes in the anabolic pathway of unsaturated fatty acids.These genes were used to detect the expression patterns of these genes in different developmental stages of tea seeds using real-time quantitative PCR;and the FAD family genes in tea leaves were tested under abiotic stress.Overexpression vectors of CsFAD6 and Δ8-CsFAD were constructed and Arabidopsis was transformed with Agrobacterium tumefaciens to obtain transgenic lines.The main results of this thesis are as follows:1.Cloning and bioinformatics analysis of CsFAD gene from tea plant11 tea plant CsFAD genes were cloned and named CsFAD2-1,CsFAD2-2,CsFAD3,CsFAD6,CsFAD7,CsFAD8,Δ6-CsFAD,Δ7-CsFAD,Δ8-CsFAD,FAB1 and FAB2.Through the MEME prediction of the conserved domain analysis,the 11 CsFAD proteins all contained typical FAD conserved domains.Phylogenetic tree analysis showed that 11 CsFAD proteins can be divided into four subfamilies:ω3/ω6-FAD,FAB,Front end FAD,and Δ7/Δ9 FAD.2.Abiotic stress response analysis of CsFAD family genes in tea plantThe expression patterns of 11 CsFAD genes in three stress treatments including low temperature,ABA and drought were studied.The results showed that under low temperature stress,the relative expression levels of CsFAD2-2,CsFAD8,Δ6-CsFAD,Δ7-CsFAD,Δ8-CsFAD,CsFADB2 increased more than 10 fold;Under ABA stress,CsFAD2-2,CsFAD8,Δ7-CsFAD,The relative expression levels ofΔ8-CsFAD and CsFADB2 increased more than 5-fold;under drought stress,the relative expression levels of CsFAD2-1,CsFAD2-2,CsFAD8 and Δ8-CsFAD increased more than 3 fold.Among them,seven genes,CsFAD2-1,CsFAD2-2,CsFAD8,Δ6-CsFAD,Δ7-CsFAD,Δ8-CsFAD,and CsFADB2,were significantly induced by these three stressors.The results showed that CsFADs played an important role in the response of tea plants to abiotic stresses.3.Analysis of the anti-stress function of CsFAD6 and Δ8-CsFAD1)The promoter sequence of Δ8-CsFAD gene was cloned to 2 040 bp.Promoter element prediction showed that there are multiple abiotic stress response elements such as LTR,MBS,and HSE stress response elements and ACE,G-Box,Several light-responsive elements such as GA-motif and several hormone-responsive elements including gibberellin,ABA,salicylic acid,and jasmonic acid,suggest that Δ8-CsFAD may be involved in many stress responses in tea plants,and they are resistant to stress in tea plants.Play a certain role.2)Through the temporal expression of CsFAD6 and Δ8-CsFAD in tobacco leaves,the results showed that CsFAD6 is located in the chloroplast and Δ8-CsFAD is located on the cell membrane.3)The CsFAD6-pH7FWG2 and Δ8-CsFAD-pH7FWG2 overexpression vectors were constructed and the target gene was overexpressed in Arabidopsis thaliana by Agrobacterium-mediated infusion method.After three generations of screening,homozygous transgenic overexpression T3 was obtained.The mustard lines lay the foundation for the subsequent study of the functions of CsFAD6 andΔ8-CsFAD in stress resistance of tea plants.4.Cloning and Expression Analysis of Key Enzymes for Fatty Acid Synthesis in Tea PlantThe key enzyme genes in 9 saturated fatty acid metabolic pathways associated with fatty acid formation in tea seed oil and the CsFAD family genes,the key enzymes in 11 unsaturated fatty acid metabolic pathways,were detected during tea seed development(August-October,August.31)The expression pattern.The results showed that the key genes for the synthesis of saturated fatty acids,CsACP,CsBC,CsHD,CsKASⅠ-2,CsKASⅢ,and CsFAT1,reached the highest expression levels during the period of tea seed development from August 1 to August 31;CsFAD2-2,Δ6/Δ7/Δ8-CsFAD showed an upward trend during the development of tea seed from August 1 to August 30,reached a maximum on August 31,and then showed a declining trend;CsFAD2-1,CsFAD3,CsFAD6,CsFAD8,FAB1 and FAB2 expression levels The maximum value was reached between October 15 and October 30 in the later stages of tea seed ripening.Combining the changes of fatty acid accumulation in oil during the development of tea seed,it was inferred that the key fatty acid synthesis key enzymes played an important role in the early stage of fatty acid formation in tea seed oil,and accumulated 16C and 18C saturated fatty acids;during the middle and late stages of tea seed development,it was not saturated.Fatty acid synthase genes FAD and FAB play a major role in catalyzing the accumulation of saturated fatty acids from 18C to the unsaturated fatty acids oleic acid and linoleic acid,resulting in the formation of highly unsaturated fatty acid tea seed oil characteristics. |
