| Saffower is the long cultural history traditional Chinese medicinal(TCM)herb in China,and used in many aspects.The content of linoleic acid(LA)is more than 70%of total fatty acid(FA)in ordinary safflower cultivars.Safflower can be classified three types according to the content of linoleic acid.The linoleic acid as essential fatty acid for human has multiple efficiency including reducing viscosity and the contents of cholesterol of blood,preventing cardiovascular and cerebrovascular diseases.Unsaturated fatty acid(UFA)is the main constituent of cell membrane and plays an important role in resisting biological and abiotic stress.The biosynthesis of unsaturated fatty acid(UFA)relies on the catalysis of fatty acid synthase system and a series of fatty acid dehydrogenase.Many key genes that control the synthesis of fatty acid dehydrogenase had been cloned and sequence analysis were executed.Although some articles have reported the biosynthesis pathway of fatty acid in recent years,the studies about the accumulation pattern of fatty acid which respond to the abiotic stress such as cold and exogenous plant hormones stress have not or partially been reported.Therefore,in this study,the plants of elongation stage were sprayed by gibberellin and paclobutrazol with different concentrations in safflower,and cold stress was executed using different cultivars with different contents of main fatty acids at bud and seeding stage.The change of contents of main fatty acids were detected by GC-MS in different tissues such as leaves and seeds.To further study the accumulation of UFA in molecular level,RTq-PCR technique were used to analyze the expression pattern of partially key genes in biosynthesis of fatty acid for different development stages of seeds.These genes are areacetyl-CoA carboxylase(ACC),acetyl-coa,Beta-ketone fatty acyl-the ACP synthetase Ⅱ(KASII),Delta 9-fatty acids to saturated enzyme(SAD),Omega-6 fatty acids to saturated enzyme(FAD2,FAD6).The main research results are as follows:1.The plants in elongation stage were sprayed by gibberellin(30、100、170 mg/L)and paclobutrazol(30、90、150 mg/L)that can inhibit the biosynthesis of gibberellin in safflower.The results showed that,treated with gibberellin the plant height and the length of branches were increased significantly but the total number of fruit ball and the number of first branch each plant were decreased significantly.Otherwise,the early flowering season ahead of the control one to four days under gibberellin stress.However,treated with paclobutrazol the length of branch were decreased significantly and the early flowering season were put off one or two days.Overall,the total compositions and contents of fatty acid of leaves and flowers were not changed significantly but the contents of oleic acid and linoleic in seeds were influenced significantly under gibberellin and paclobutrazol stress.The contents of linoleic in seeds of safflower was 67.83%after spraying gibberellin,and 5.25%was higher than the control samples under the gibberellin stress with the concentration 30 mg/L.However,the content of oleic was 20.05%and 4.81%higher than the control under the paclobutrazol stress with the concentration 150 mg/L.The O/L value was improved significantly in seeds collected at 20 days after flowering after spraying paclobutrazol.The expression level of the key enzyme genes that control biosynthesis of fatty acid were detected and analyzed by Q-PCR for the seeds stressed by exogenous hormone.The results showed that except CtFAD2-3 and CtFAD2-6 were up-regulated at 10 days after flower(DAF)and 15 DAF,other genes were nearly expressed stably at seed different development stages.After 30 mg/L gibberellin stress,the accumulations of mRNA of each gene were increased compared with control,but all genes expression level were lower at 5 DAF.However,after 10 days,the expressions of CtKASⅡ、CtSAD、CtFAD2-1 were increased significantly,others were decreased in seeds.The expressions of all genes were lower than other stages at 15 DAF significantly.After 20 days,the expressions of CtKASII、CtFAD2-8,CtSAD CtFAD2-1 were higher than the expression after 10 days.For the samples stressed by 150 mg/L paclobutrazol,the expressions of all genes were lower than other stages at 0 DAF and 5 DAF.After 10 days,the expression of CtSAD and CtFAD2-1 were increased and the CtKASⅡ was changed not significantly.After 20 days,the accumulations of mRNA of each gene were increased.2.Four different temperature(4℃、10℃、16℃、24℃)were executed for safflower seeding after two months.Different tissues including root,steam and leaves were collected at 0 h,1 d,2 d,4 d and 12 d after stress.The compositions and contents were detected by GC-MS.The results showed that the contents of linoleic and linolenic acid increased gradually but were decreased after 4 days and the content of palmitic acid decreased under cold stress with the temperature 4℃ and10℃ for the safflower plants that have lower linoleic acid level.However,two UFA fastly decreased under 16 ℃ and 24 ℃ conditions.The SFA all increased under any temperature condition.On the contrary,for higher linoleic acid cultivars,the contents of linoleic and linolenic acid increased under 4 ℃,and increased at 0 h-2 d under 10 ℃.Under 16 ℃ and 24 ℃,the content of two UFA decreased gradually and palmitic acid increased.Stearic acid improved under four different temperatures conditions.The contents of fatty acid in roots and steams were changed significantly under cold stress.The results indicated that the accumulation of UFA increased under cold stress,and SFA can be improved under high temperature conditions.The compositions and contents of fatty acids were changed differently in different safflower cultivars and different tissues.3.Cold stress was executed under 12 ℃ at bud stage for different safflower cultivars that have different contents of linoleic.The results showed that the contents of UFA for PI 401477 changed not in leaves,but increased the content of linolenic acid of PI470942,decreased the content of linolenic acid,increased the content of palmitic acid of PI544021.In flowers,the contents of linoleic acid decreased and palmitic acid increased slightly for medium cultivars.The linolenic acid improved significantly for PI 470942.For PI 544021,the contents of linoleic acid and linolenic acid were all improved.In seeds,the contents of linoleic acid and oleic acid were improved for medium cultivars.In seeds,treated by low temperature the content of linoleic acid and oleic acid increased in PI401477;decreased the content of linoleic acid significantly,increased the content of oleic acid in PI470942;decrease the content of linoleic acid of 20～25 d seed,increased the oleic acid in PI544021.SAD synthesis oleic acid,FAD2 synthesis linoliic acid.CtFAD2-1 is the key gene that synthesis linoleic acid in seeds.Measuring the expression levels of the key genes by QPCR.The results showed that different expression pattern of selected key genes emerged in different cultivars.In the control,the expression level of FAD2-3 and FAD2-6 is high in PI401477.Besides FAD2-3 and FAD2-6,the expression level of FAD2-4 and FAD2-5 is high relatively in PI470942;In PI544021,the expression level of FAD2-1 is very high in 25 d sees differently.For medium cultivar,the expression levels of most of key enzyme genes were improved significantly under cold stress.CtFAD2-2,CtFAD2-3,CtFAD2-6 have higher expression levels.The accumulation of mRNA of CtFAD2-2,CtFAD2-6 and CtFAD2-8 improved in 20 d seeds.The expression of CtKASⅡ、CtSAD and CtFAD2-1 were hardly detected.For cultivar with lower linoleic level,the expression of genes were higher than control after initial stress.After 10 days,depressed expression levels were detected except gene CtFAD2-8.After 20 days,the changes of expression quantity between stress samples and control samples were not significant.For higher contents of linoleic cultivar,the accumulation of mRNA of genes were all increased except CtACC at initial stage after stress but decreased after 10 days in seeds.The expression of genes were higher other stages,especially the expression of CtKASII,CtFAD2-1 and CtFAD2-8 increased significantly.The above results indicated that the compositions and contents of fatty acids changed differently in different safflower cultivars and different tissues and the accumulation of UFA increased under cold stress,and SFA can be improved under high temperature conditions.The content of fatty acid is relative to the expression level of key genes,esprcialy the CtFAD2-l and CtSAD.Different materials responsed differently treated by low temperature,through different ways resisting low temperature,it is better for us to further study the mechanism to low temperature in safflower. |
