Genetic Transformation Of Two Lipids Biosynthesis Genes Into Brassica Napus L

As one of the four major oil crops in the world,rapeseed is not only the main source of edible vegetable oil,but also an important source of feed protein and an ideal raw material for biodiesel.Elevation of seed oil content and Improvement of fatty acid composition in seed oil are the important goals of B.napus genetic breeding.Diacylglyceryl transferase gene（DGAT1）plays an important role in seed lipids accumulation.The oleic acid desaturase gene（FAD2）can be used to genetically regulate oil composition.Present works have cloned BnDGAT1 gene and BnFAD2,and transformated these two genes into oilseed rape to to improve oil content and change the fatty acid composition of rape seeds,new materials were obtained.The main results are as follows:1.Genetic transformation of BnDGAT1（1）Gene cloning:One of 3 copies of the DGAT1 gene in B.napus L.BnDGAT1-1 was cloned from double-low variety Zhongshuang 11（ZS11）for functional analysis and genetic transformation.This gene has 16 exons and 15 introns,with ORF of 1506 bp in length,and has a conserved domain characteristic of diacylglyceryl transferase.（2）Genetic transformation of BnDGAT1-1:Overexpression vector of BnDGAT1-1 was constructed under seed-specific promoter Napin in replace of the CaMV35S promoter.With double-low variety ZS11 and NY9808 as recipients,transformation with this construct resulted in 4 and 3 transgenic lines,respectively.The gene expression of BnDGAT1-1 significantly increased in the transformants silique,and the seed oil content in seeds of transformants was also significantly increased by 2-8%.2.Genetic transformation of BnFAD2（BnC05g40970D）（1）Gene cloning:One of 3 copies of the fatty acid desaturases genes（BnFAD2）in B.napus was cloned from ZS11.The similarity to BnC05g40970D is 100%and that to BnA05g26900D is 96.88%.This gene has 3 exons and 2 introns,with ORF of 1154 bp in length,and has a conserved domain characteristic of fatty acid desaturase.（2）Construction of overexpression vector and the results of genetic transformation:Overexpression vector of BnFAD2 was constructed under seed-specific promoter Napin in replace of the CaMV35S promoter.The ZS11 and NY9808 were used as recipients.Agrobacterium-mediated method was adopted.2 and 5 T1 transgenic plants were harvested,respectively.This gene expression level in the transformed siliques was increased by 1-4 times.3.Gene editing of BnFAD2（1）Construction of vector for gene editing:Two Targe spots targets(TS1 and TS2 were designed within the conserved region of the gene,with a distance of about 220 bp.The GC content of TS1 and TS2 was 60%and 55%,respectively,beneficial to target editing.To construct sgRNA expression box,AtU3b and AtU3d promoter promoter were selected for TS1 and TS2,recpectively.（2）Genetic transformation:By use of the CRISPR/Cas9 gene edtiting system,three recipients were transformed.With specific primers designed on Cas9p sequence 5,5 and 4 T1 generation transgenic plants were obtained,respectively for ZS11,NY9808 and CE001.（3）The gene-edited plant identification:By use of the gene sequences of BnA05g26900D and BnC05g40970D,gene-specific primers were designed and used to detect the gene-edited plants in T2 generation,gene sequencing results shown that 4 editing plants were obtained in the rapeseed line CE001.