Analysis Of Differences In Fatty Acid Composition And Gene Expression Profiles During Cottonseed Development

Object:Cotton is not only the most important fiber crop,but also an important oil crop.Cotton seeds contain 25%to 35%oil,which is the fifth largest edible vegetable oil in China.As an important source of edible vegetable oil,the fatty acid composition and nutritional function of cottonseed oil are attracting more and more attention.It would be beneficial to improve the quality of cottonseed oil to explore the proportion of different fatty acids and their correlations and the expression and regulation of related genes in cotton seed development.The method of fatty acid methyl esterification of rapid quantitative of fatty acid compisitions were proposed.Dynamic change of the fatty acid components and gene expression profiles in the development of cottonseed would be analyzed.The results would provide a scientific basis for improving the quality of cottonseed oil.Methods:The fatty acid composition in cottonseed of“Xinluzao 39”and“Xinluzao 50”methylated by three kinds of catalysts,KOH-methanol method,H₂SO₄-methanol and 14%BF₃-methanol methods,was determined by GC-MS.The quantitative standard curve was established by mixing 37 fatty acid methyl esters determinated by GC-MS,and the external standard method was used for quantitative determination.The fatty acid composition of 12different developmental stages at 5-60 DAF of cottonseed from cotton variety"XinLuZao 33"were measured by GC-MS.The dynamic changes of fatty acid components and their correlation in the development of fatty acid in cottonseed were analyzed.Using RNA-Seq technology,the gene expression profiles of cottonseeds at different developmental stages were analyzed by digital gene expression profiling,and the correlations between fatty acid components and related gene expressions were analyzed.Results and Conclusion:1.The fatty acid composition in cottonseed of“Xinluzao 39”and“Xinluzao 50”methylated by three kinds of catalysts,KOH-methanol method,H₂SO₄-methanol and 14%BF₃-methanol methods,was determined by GC-MS.The results showed that there are 10 kinds of fatty acid composition in cottonseed oils,which could be detected on GC-MS analysis by the esterification of fatty acids with KOH-methanol.Only 6 kinds of fatty acid composition could be detected by H₂SO₄-methanol and 14%BF₃-methanol method.The oil contained high level of linoleic acid,polyunsaturated fatty acid,followed by saturated fatty acid,palmitic acid and monounsaturated fatty acid,oleic acid.The linoleic acid content per gram cottonseed was 147.29 mg and 163.2 mg in seed oil of Xinluzao 39 and Xinluzao 50,respectively,which accounted for 60.12%and60.23%of its oil content.The oil also contained high level of palmitic acid,contents of which were 65.05 mg and 62.28 mg,accounting for 26.55%and 23.24%of their oil content respectively.The content of oleic acid was 24.79 mg and 22.19 mg,accounting for 8.28%and 10.12%of their oil content respectively.2.There are 13 kinds of fatty acid composition in cottonseed oils,which could be detected by GC-MS analysis in the 5DAF cottonseed.At this stage,The content of palmitic acid（C16:0）is a relatively high and reached to 33.96%.The relative content of stearic acid（C18:0）is 9.02%,and the relative content of monounsaturated oleic acid（C18:1）is 23.42%.The contents of unsaturated linoleic acid（C18:2）and linolenic acid（C18:3）were 20.30%and 7.44%,respectively.At the seed ripening stage（60 DAF）,the relative contents of fatty acid components C16:0,C18:0,C18:1,C18:2,and C18:3 were 22.35%,2.31%,15.59%,57.62%,and 0.02%,respectively.The results showed that palmitic acid（C16:0）,stearic acid（C18:0）and linolenic acid（C18:3）accumulated mainly during the early-middle stage of seed development.But oleic acid（C18:1）,linoleic acid（C18:2）accumulated mainly during the middle-late stage of seed development.With the increase of content C18:1 and C18:2,the remaining fatty acids showed a significant decline.C18:1 and C18:2 synchronously accumulated,and there was significantly positive correlation between the C18:1 and C18:2,negatively correlated with C18:3 and C16:0.With an increase in the content of C18:1,the C18:2 content also increases,while at the same time it leads to a decrease in the C18:3 content.In the process of cotton seed development,C18:1 and C18:2 are the main factors affecting the fatty acid content of cotton.3.The results of gene expression profiling analysis showed that the number of differentially expressed genes was the highest at 5 DAF,and 55 DAF and 60 DAF.Compared with 0 DAF,the number of up-regulated genes was 5709 and down-regulated genes was 6454 at 5 DAF.Compared with 55 DAF,the number of up-regulated genes was 5169 and down-regulated genes was 4256 at60 DAF.The number of gene differences is relatively less during the middle stage of seed development.Cluster analysis by differential gene showed that there are more up-regulated genes at 5²5 DAF,and more down-regulated genes at 30⁶0 DAF.By differential gene GO enrichment analysis and Pathway enrichment classification analysis,we can find that in the early development of cottonseeds involves fatty acid biosynthesis,fatty acid metabolism and keratin and wax biosynthesis.4.In the early stage of cottonseed development,when the accumulation of C16:0 and C18:0exceeds a certain threshold,FAD2 gene expression is induced to increase,resulting in massive C18:2 synthesis.At the 20DAF,the expression of FAD2 gene was significantly increased,which resulted in the significant increase of linoleic acid（C18:2）content at 20 DAF.At 40 DAF,the expression of FAD2 gene reached to the maximum.The content of linolenic acid（C18:3）changed mainly in the early stage of cottonseed development,and the content of linolenic acid decreased during the early stage of 5DAF¹5DAF.The analysis of gene expression profiles in the development of cottonseed showed that the FAD3 gene was expressed at the high level of 5DAF and then decreased significantly.At 25 DAF,the FAD3 gene level was still significantly decreased,and the level of expression remained low.In general,the gene expression level of key enzymes of fatty acid synthesis plays an important role in regulating the content of fatty acid components during the development of cottonseed.The form of various fatty acid accumulation patterns in plants is closely related to the plant’s self-defense and protection mechanisms for offspring.The interaction and relationship between fatty acid metabolism-related gene expression and fatty acid accumulation remains to be further studied.