| Rapeseed is an important edible oil crop in the world.China currently consumes about6.1 million tons of rapeseed oil annually,accounting for about 20% of the consumption of edible vegetable oil.The average rapeseed oil content is about 42%,containing 7% of saturated fatty acids(16:0 and 18:0),about 60% of monounsaturated fatty acids(18:1),and about 33% of polyunsaturated fatty acids(18:2 and 18:3).Polyunsaturated fatty acids have high nutritional value,but high levels of polyunsaturated fatty acids make rapeseed oil easily oxidized and rancid,producing volatile and unpleasant harmful substances,thereby reducing the nutritional and commercial properties of rapeseed oil.Oleic acid has high nutritional value and has a health-care effect on the cardiovascular system,which can reduce the incidence of heart disease and arteriosclerosis.High-oleic rapeseed oil is not easily oxidized and rancid,and its commerciality is higher than that of common rapeseed oil.Therefore,to improve rapeseed oil content and oleic acid content is an important research direction of rapeseed research.Acyl-ACP thioesterase is an important enzyme in fatty acid synthesis.The fatty acids synthesized in the plastid are hydrolyzed to free fatty acyl-CoA and ACP by their action,thereby ending the elongation of fatty acid carbon chain.There are two families of thioesterase genes,fatA and fatB,in rape,and there are multiple members in each gene family.Fatty acid-ACP sulfatase not only affects the level of oil in seeds,but also affects the fatty acid composition in seeds.Therefore,it is of great significance to study the fat acyl-ACP sulfatase gene(fat)for the study of oil content and fatty acid composition.In this study,we analyzed the RNA-seq data of Brassica napus with different oil content and oleic acid content and screened out 3 fatA and 2 fatB genes with significant differences.Using RT-qPCR methods,the expression levels of these five thioesterase genes at different developmental stages of different rapeseed oil content and oleic acid content were analyzed.Through molecular cloning,the full-length CDS of these five genes were obtained,and then These fragments were inserted into the yeast expression vector pYES2.0 to construct a yeast expression vector and yeast expression.The oil content and fatty acid composition of the transgenic yeast were analyzed.Based on the results of the analysis,the plant expression vectors PBI121-fatA1 and PBI121-fatA3 were constructed by selecting two genes(fatA1and fatA3)that contribute significantly to oleic acid,and wild type Arabidopsis thaliana plants were transformed using Agrobacterium-mediated transformation.The main findings are as follows:1.Through molecular cloning,five full-length CDS of fatA and fatB genes were obtained.By RT-qPCR analysis,the expression trends and regularities of the five thioesterase genes in different seed development stages of oilseed rape with different oil content and oleic acid content were basically clearly defined.2.Analyze the fatty acid composition of the yeast gene with fat by gas chromatography.The results showed that the contents of stearic acid(C18:0)and oleic acid(C18:1)in the transgenic yeast were increased compared with the control.Among them,the oleic acid content of the fatA1 gene was the highest.3.Soxhlet extraction method was used to determine the oil content of the yeast gene.The results showed that the oil content of all transgenic yeasts had a certain increase compared to the control.The oil content of yeasts with fatA1,fatA2,fatA3,fatB1 and fatB2 genes increased by87.806%?6.428%?36.05%?16.398 %and 24.917% respectively.4.Infection of Arabidopsis thaliana by Agrobacterium-mediated method was successfully screened for positive T1 transgenic Arabidopsis seedlings.Based on the experimental results,we believe that the function of fatA1 gene is the strongest among the five fatty acyl-ACP sulfatase genes,and it contributes most to the rape oil content and oleic acid content.This study provided a new scientific basis for the high oil content and high oleic acid breeding of rapeseed. |
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