Differences Of Oil Accumulation And Key Gene Expression During The Seed Development In Brassica Napus L.

Rapeseed is one of the most important oil crops in the world.Its oil content is an important economic tarit and a key factor in determining the yield and the quality of rapeseed.Rapeseed oil is rich in fatty acids and vitamins,which is beneficial to human physical and mental health,so it’s a very high-quality edible oil.Due to the complex genetic characteristics of rapeseed oil content（a typical quantitative trait controlled by main gene+multi-gene）,the seed oil content is regulated by genetic development and influented by environmental factors.As a result,the rapeseed oil is not enough to meet the growing demand of edible oil.High oil content rapeseed varieties has always been one of the important targets of rapeseed breeding.In this study,5 high/low oil content lines of Brassica napus L.with different genetic backgrounds were used as experiment materials.By measuring the oil content,main fatty acid content,soluble protein content and soluble sugar content of seed in the development process of rapeseed,the relationship was explored between the related substances synthesized in seed and silique wall and the oil content accumulation.Moreover,transcriptome sequencing was performed on 30d,40d,50d seeds and 30d silique wall to reveal the key genes regulating oil accumulation in Brassica napus seeds at the molecular level.The purpose was to investigate the causes of the differences in different oil content rapeseed lines,to deepen the understanding of oil accumulation and regulation network of rapeseed,to lay the theoretical foundation and provide practical guidance for the breeding of high oil content rapeseed varieties.The main results were as follows:1.Dynamic analysis of oil and main fatty acids accumulation in high/low oil content linesDuring the seed development,the modes of oil accumulation in high/low oil content lines were basically same:only a small amount oil accumulated in 0d-20d after flowering,in 20d-30d oil accumulation was relatively slow,30d-40d was a rapid increase period of oil accumulation,40d oil content accounts for 70.11%-93.45%of the mature seed oil content.After 40d,low oil content（LOC）lines entered low-speed accumulation,while the high oil content（HOC）lines still maintained a high increased rate until reaching oil content peak at 50d.The main fatty acid compositions and their accumulation patterns were consistent,with the highest oleic acid content,followed by linoleic acid,linolenic acid,palmitic acid and stearic acid,and had low content of eicosenoic acid and erucic acid.The saturated and polyunsaturated fatty acids were mainly accumulated in the early stage of seed development,and the oleic acid content increased with the development of seed.The proportions of main fatty acids in different materials maturity seed were similar:67.41%oleic acid,18.63%linoleic acid,7.27%linolenic acid,4.57%palmitic acid,and 2.11%stearic acid.The conversion ratios of the5 main fatty acids were less affected by the variety.2.The analysis of difference in soluble protein and soluble sugar accumulation in high/low oil content linesThe accumulation mode of seed soluble protein was in two ways,one was the soluble protein content increasing with the development of seed,and another was decreasing after increasing to the peak.The soluble protein content of the LOC lines A1341 and A1342 at 20d accounted for 70.98%and 75.92%of the peak.The protein synthesis of the LOC lines were earlier and more than those of the HOC lines.The accumulation patterns of soluble sugar in seed and silique wall were different.The trend of soluble sugar content accumulated in silique wall was similar between HOC and LOC lines,there was a large fluctuation from 20d to 30d,the soluble sugar content showed high or low peak,and then continuously decreased with seed development process.It was speculated that the photosynthesis and transshipment capacity of sillique wall was the strongest before 30d.However,there was a big difference in the accumulation trend of soluble sugar content in seed,and it was always lower in seed of HOC lines than that of LOC lines during seed development.The seed with high oil content had stronger metabolic capacity and the more sugars may be used in the oil formation.3.Transcriptome sequencing analysis of 30d,40d and 50d seed in high/low oil content lineTranscriptome sequencing was performed on the seeds of HOC A517（47.30%）and LOC A1342（36.21%）.There were many differential genes at different period in each line,and fewer differential genes between the two lines in the same period and the number was similar.GO and KEGG enrichment analysis results of all differential genes showed that differential genes mainly involved in cell components,followed by biological processes,with the least molecular function,and significantly enriched in carbon metabolism,amino acid biosynthesis,starch and sucrose metabolism,phenylpropane biosynthesis and other related pathways.Afterwards,the co-expression trend analysis of high/low oil content lines respectively found that there were up and down trends of differential genes.Among them,down-regulated genes in LOC line were mainly enriched in fatty acid synthesis pathways such as acetyl-Co A metabolism and positive regulation of fatty acid biosynthesis.Up-regulated genes in HOC line were mainly enriched in malate synthase activity,glyoxylate cycle,fatty acidβ-oxidation and other pathways related to fatty acid degradation.The results indicated that the fatty acid synthesis intensity decreased with the seed development,but fatty acid degradation continued to increase activity.WGCNA analysis showed that the glucose catabolism of HOC line was significantly stronger in 40d and 50d after flowering,and expression levels of most genes in gluconeogenesis pathway were also higher in 30d,40d and 50d after flowering.Further analysis of genes encoding key enzymes and transcription factors for fatty acid synthesis and degradation revealed that most of the enzyme genes related to fatty acid synthesis were down-regulated with the seed development,while the genes related to fatty acid degradation were up-regulated.The expression levels of 7 key enzymes encoding FAS,FATA and DGAT in fatty acid synthesis pathway and KAT,MLS,ACO and ICL in fatty acid degradation pathway were significantly higher than those of other enzyme genes.Among them,Bn FAS,Bn KAT and Bn ACO had no significant difference in high/low oil content lines.Bn FATA1-1,Bn FATA1-2,Bn FATA1-3,Bn FATA2-1,Bn FATA2-2 and Bn DGAT1-2,Bn DGAT2,Bn DGAT3 had higher expression levels in high oil content line,while the expression levels of Bn MLS-1,Bn ICL-1,Bn ICL-3 and Bn ICL-4 in low oil content line were significantly higher than those in high oil content line.It could be seen that the fatty acid synthesis pathway was more active,and the degradation pathway was relatively weak in the high oil content line.FATA,DGAT,MLS,and ICL were the most key genes that caused the difference between high and low oil content.With seed maturation,the expression levels of genes encoding transcription factors gradually decreased,so the positive regulation of oil synthesis gradually weakened.It was worth noting that the expression trend of ABI3 peaked at 50d,and it played a major positive role in the seed development and the oil synthesis in the late stage of seed development.4.Transcriptome sequencing analysis of 30d silique wall in high/low oil content linesBy analysising transcriptome sequencing of 30d silique wall,the results showed that the differential genes were significantly enriched in sugar metabolism,carotenoid biosynthesis.Furthermore,to obtain the detailed distribution of the differential genes in the photosynthetic pathway,the log₂FC value of the differential genes expression was used to draw the map of the photosynthetic pathway in Map Man.It was found that the genes involved in light response were more active in HOC line,and 199 of 255differential genes were up-regulated.Among the genes related to sucrose transporting,18 genes encoding the SWEET transporter,16 genes were highly expressed in HOC line.4 genes encoding SUC,except for Bna C05g17970D,which was highly expressed in HOC line,the other genes expression were no difference or even lower than LOC line.This result revealed that HOC lines may had higher photosynthetic efficiency,while high/low oil content lines had no significant difference in sucrose transport capacity,and further experiments were needed to verify it.