Function Of BnaLPAT2 Gene In Brassica Napus

Rape is the most widely sown area and the highest total output of oil crops in China.The demand for rapeseed oil accounts for more than 1/2 of the total vegetable edible oil in China,and the rapeseed planting industry has become an important industry to promote the safe development of edible oil industry in China.It has been proved that rapeseed yield can be increased by 2.5%when the oil content in rapeseed oil is increased by1%.Improving oil content and oil quality in rapeseed seeds has become an important direction in rapeseed breeding in China.Lysophosphatidic acid acyltransferase（LPAT）is one of the important rate-limiting enzymes in plant biosebum synthesis.It has the biological function of regulating fatty acid chain assembly of triglycerides,thus affecting fatty acid composition and oil content in rape seeds.In this experiment,The T₀generation of transgenic rapeseed was identified by PCR technology,and the transgenic T₃generation of rapeseed was cultured in an artificial climate box.In this study,PCR positive detection efficiency,CRISPR/Cas9 target editing,BnaLPAT2 gene expression pattern in tissues of transgenic and wild-type rapeseed,fatty acid components and oil content in transgenic rapeseed seeds were studied.1.The expression of BnaLPAT2 gene in the tissues extracted from"Zhongshuang 6"at flowering stage was the lowest in roots and the highest in flowers.After self-pollination,the expression of BnaLPAT2 gene increased first,then decreased and then increased with the development of silipod,and reached the highest value on 35 day of silipod development.The expression of BnaLPAT2 gene was the lowest in stem and the highest in flower of"Xiangyou15".After self-pollination,the expression of BnaLPAT2 gene increased first,then decreased,then increased and then decreased slowly with the development of silipod.The expression of BnaLPAT2 gene reached the highest value at 28 day of silipod development.2.PCR was used to detect transgenic rapeseed T₀generation plants,and 18 rapeseed plants with BnaLPAT2-A07 gene overexpressed by 35S promoter were obtained,among which 15 were PCR positive,with a positive detection rate of 83.33%.BnaLPAT2-A07 gene was overexpressed in 16 rapeseed plants driven by the seed-specific promoter Napin,of which 11 were PCR positive and the PCR positive detection rate was 68.75%.Real-time quantitative PCR detected that the expression level of BnaLPAT2-A07 gene in T₃generation of overexpressed Brassica napus.was different from that of wild type Brassica napus,and the 35S promoter driven the expression level of BnaLPAT2-A07 gene in all tissues of transgenic Brassica napus.was significantly increased.Napin promoter specifically drives the expression of BnaLPAT2-A07 gene in transgenic brassica napus scones.The content of palmitic acid and linolenic acid in oilseed T₃was significantly increased and the content of oleic acid and linoleic acid in oilseed T₃was decreased by 35S promoter driving BnaLPAT2-A07.The content of linolenic acid increased by 3.13%,palmitic acid increased by0.17%,oleic acid decreased by 1.29%,linoleic acid decreased by 1.91%,and oil content of transgenic rapeseed seed increased by 1.39%compared with wild type rapeseed seed.The contents of linoleic acid and linolenic acid of Napin promoter driving BnaLPAT2-A07 gene were increased and the contents of oleic acid and stearic acid were decreased compared with the control.Linoleic acid increased by 1.77%,linolenic acid increased by 1.47%,oleic acid decreased by 2.78%,stearic acid decreased by 0.39%,and oil content of transgenic rapeseed seeds increased by 2.36%compared with wild type rapeseed.、3.Eighteen CRISPR/Cas9 edited rapeseed strains were obtained by PCR detection technology,among which 10 were PCR positive,with a PCR positive rate of 55.56%.Sanger sequencing method detected that BnaLPAT2 gene was edited in 5 strains of 18 T₀generation rapeseed,and the gene editing efficiency was 27.78%.The editing efficiencies of BnaLPAT2-A04、BnaLPAT2-A07、BnaLPAT2-C08 and BnaLPAT-2-A09 gene targets were22.22%,27.78%,11.11%and 5.56%,Respectively.The editing efficiency of sg RNA 1 and sg RNA 2 is 9.72%and 12.50%.The editing types produced by gene editing included deletion,substitution,insertion and heterozygous mutation,accounting for 41.67%,15.72%,10.72%and 31.91%of the total mutations,respectively.The editing types of rapesus napus at T₀generation were deletion>heterozygous mutation>substitution>insertion.Sequencing of 401-1,401-2 and 401-3 brassica napus in T₁generation showed that the editing type of BnaLPAT2 gene target was similar to that in T₀generation,which proved that gene editing could be effectively passed on to future generations.The expression levels of CRISPR/Cas9 edited BnaLPAT2 gene homologous copies in different rapeseed lines decreased in different tissues compared with the control rapeseed Xiangyou 15.The fatty acid composition and oil content of CRISPR/Cas9 edited rapeseed T3 seeds were detected.The oleic acid content of CRISPR/Cas9 edited rapeseed T₃seeds was significantly higher than that of control rapeseed,while the linolenic acid and stearic acid contents were significantly lower.The oleic acid content increased by 2.69%,the linolenic acid content decreased by 1.13%,and the stearic acid content decreased by 0.86%,and the oil content of edited rapeseed seed decreased by 2.51%compared with that of wild type rapeseed.