The Editing Of Chlorophyll Synthase And Seed-Specific Overexpression Of ATHPT In Brassica Napus

Vitamin E（including tocopherols and tocotrienols）is a kind of vitamin and it is of great significance to humans and animals..They itself cann’t synthesize vitamin E but only rely on dietary supplements.Rapeseed（Brassica napus L,AACC,2n=38）is one of the most important oil crops and an important source of human supplementation.Breeding rapeseed varieties with high vitamin E is one of the main objectives of crop breeding research.Previous studies showed that during the synthesis of tocopherols in model plant Arabidopsis thaliana,Homogentisate Phytyl Transferase（HPT）is the rate-limiting enzyme,Chlorophyll Synthetase（CHLSYN）competes with HPT for the same substrate PDP,so we named it“negative regulator”.CRISPR/Cas9 is a novel gene editing technology that can simultaneously edit multiple genes in a genome.In this study,CRISPR/Cas9 were used to knock out the different copies of Bn CHLSYN,as an important foundation for the establishment of germplasm resources with high vitamin E content,at the same time,for further increase the vitamin E content in rapeseed,we also specifically overexpressed At HPT.The main results are as follows:1.Bioinformatics analysis of CHLSYN shows that in Brassica napus there are four homologous:Bna C1CHLSYN,Bna A1CHLSYN,Bna C8CHLSYN and Bna A9CHLSYN,The homology of them is very high,Bna C1CHLSYN and Bna A1CHLSYN is 95%,Bna C8CHLSYN and Bna A9CHLSYN is 91%.2.Real-time PCR was used to detect the expression level of four copies of chlorophyll synthetase in leaves of different parts of plants in J9707,and the seeds in different developmental stages.The results showed that the relative expression of Bna C1CHLSYN is highest in leaves and seeds.For Bna A9CHLSYN,the relative expression in leaves is the lowest,inferior than Bna C1CHLSYN in seeds.3.CRISPR/Cas9 was used to design the specific sg RNA to knockout Bna A9CHLSYN、Bna A9CHLSYN and Bna C8CHLSYN,four copies of Bn CHLSYN These three vectors were transformed into the hypocotyls of J9707 using Agrobacterium mediated genetic transformation,after testing the T₀ plant,the number of editting plants which aims at one or two copies of Bna A9CHLSYN and Bna C8CHLSYN were 11 and16,respectively.However,the vector of knocking out four copies of Bn CHLSYN is failed to edit at the target sites;for the two successfully edited vectors,there are there mutation types such as homozygous,heterozygous and biallelic in T₀generation,of which the highest proportion is the heterozygous mutations.In addition,there are single-base insertions,single bases or large fragment deletions,replacement,a few are complex mutations.After the selfing of the editing plants,the plants without T-DNA insertion and successful editing were successfully isolated.Some T₁ generation showed different editing types from the T₀ generation.4.Five sg RNAs were designed to aim at Bna A1CHLSYN and Bna C1CHLSYN or knocked out four copies of Bn CHLSYN.These two vectors were transformed into the hypocotyl of J9707 by Agrobacterium mediated genetic transformation.Induction of callus progeny and rooting culture have been finished.5.Using seed-specific promoter:Beta Conglycinin Promoter（βcon Pro）to construct:βcon Pro:At HPT-Red3 andβcon Pro:At HPT-1305.The tocopherol content in T₁ generation seeds were detected by HPLC.The tocopherol content in T₁ generation up to 638.7μg/g compared with J9707,2.76-fold higher than J9707.