| The synthesis of plant oils requires a large amount of NADPH to participate.Nicotinamide adenine dinucleotide phosphate(NADPH)is a reduced coenzyme II,and NADP~+is the oxidative state of NADPH.One of the important sources of NADPH is the pentose phosphate pathway(PPP).Research has found that the second step of the oxidative pentose phosphate pathway(OPPP)in Arabidopsis is is catalyzed by the PGL3 gene,a dual targeting Arabidopsis 6-phosphogluconate lactone enzyme(6-PGL).Knocking out the PGL3 gene can lead to embryonic development arrest in Arabidopsis,and there is currently little research on the function of this gene in rapeseed.This study focuses on Brassica napus as the research object,using Brassica napus J9707 as the host for rapeseed transgenic material.For overexpression,we used three types of promoters:seed specific(GLY),constitutive(UBI),and pod specific(P100)promoters,to drive the expression of AtPGL3 gene in J9707.We also used CRISPR/Cas9 technology to knock-out different copies of BnaPGL3 gene in Brassica napus.The main research results are as follows:1.Through bioinformatics analysis,we found there are four homologs of the AtPGL3 gene in B.napus genome,out of which only two were expressed(BnaA06g26920D,BnaC07g49660D).Since they are on A06 and C07 chromosomes,respectively,we named them as ja6 and jc7.2.We used CRISPR-Cas9 technology to generate single and double mutants for the ja6 and jc7 copies of BnaPGL3 genes in J9707 background.After rapeseed genetic transformation and positive selection of T0 plants,we identified homozygous single mutant for ja6 or jc7,as well as homozygous double mutants for both copies.The fatty acid composition of mature seeds in these mutants were no different from that in J9707,however the seeds of homozygous double mutant were not plump.3.We used three types of promoters-seed specific(GLY),constitutive(UBI),and pod specific(P100),to drive the ectopic expression of AtPGL3 gene in B.napus J9707.We found that seeds from transgenic materials showed no significant difference in the fatty acid composition at different developlent stages,the field phenotypes were also similar.Howver,the content of NADP~+and NADPH in transgenic seeds were generally higher than control,while the protein contents were decreased compared to control.The UBI-B6-1 transgenic line had an 11.94%increase in seed oil content compared to J9707.In summary,this study successfully created a BnaPGL3 gene mutant in Brassica napus,studied the mechanism of PGL3 gene regulating seed oil synthesis,and preliminarily explored the contribution of this gene to improving seed oil content in Brassica napus.Our study provides new genetic resources for rapeseed high oil breeding studies. |
