The Role Of Chlorophyll Synthetase For Tocopherol Synthesis In Canola And Arabidopsis Seeds

Vitamin E,including tocopherols and tocotrienols,is one of the most important fat-soluble antioxidants,essential for human nutrition and health.The demand for vitamin E is huge,about 90% of Vitamin E is artificial synthesis.But synthetic vitamin E and natural vitamin E are very different in biological activity.Therefore,it is particularly important to improve the vitamin E content of green plants,especially in oil crops.Rapeseed(Brassica napus,AACC,2n=38)is one of the most important oilseed crops worldwide.The rapeseed is a rich source of vitamin E.Increasing seed vitamin E content is one of the main objective of rapeseed breeding programs.Genome-wide association analysis(GWAS)showed that,The BnA9 CHLSYN site had a strong effect on the content of tocopherols in rapeseeds.Meanwhile,it was speculated that the "bottleneck" of the synthesis of tocopherol in seeds of rapeseed is that the chlorophyll synthase "recycle" the substrate PDP in the synthesis of tocopherol to re-synthesis chlorophyll,resulting in the low concentration of PDP.To break this "bottleneck",it is crucial to unravel the relationship between the expression level of the chlorophyll synthase gene and the synthesis of tocopherols.In this study,we use the model plan Arabidopsis to explore the relationship between the expression level of CHLSYN and the synthesis of tocopherols.The main results are as follows:1.The effect of BnA9 CHLSYN loci on tocopherol content varied among the different rapeseed varieties,based on these results we could identify two groups of rapeseed varieties,namely H-group and L-group respectively.qPCR results show different expression levels of BnA9 CHLSYN gene among the two groups.The expression level of BnA9 CHLSYN in the H-group seed was lower than that in the L-group,the tocopherol content was instead.2.Under wild type background,the expression of RNA interference(RNAi)sequences of CHLSYN in Arabidopsis,increased the tocopherol content in transgenic seed by 33.69%.However,the seed specific overexpression of CHLSYN in Arabidopsisresulted in the reduction of tocopherol content in transgenic seed by 44.73%.In other words,the expression level of CHLSYN in seeds was negatively correlated with the content of tocopherol.Similar conclusions were also obtained in Arabidopsis homozygous vte5 mutant backgrounds.3.The same RNA interference(RNAi)sequences was used to knockdown CHLSYN in Arabidopsis seeds while overexpressing At HPT,the tocopherol content in the seeds could only increase by 125.38%,while the specific overexpression of only At HPT in the seeds could increase the tocopherol content by 123.56%,hence the difference was not significant suggesting a negative effect of the promoters.4.Using a different promoter to overexpress AtHPT at the same time the RNA interference sequence tocopherol content in the Arabidopsis seeds could be increased by161.71%.While the use of the new seed specific promoter for AtHPT overexpression alone can only increase the tocopherol content by 95.99%.