Creating Cotton Germplasm With High Oil And Low Phenol By Exogenous DGAT And GoPGF Gene Editing

Cotton is a major fiber and an important oil crop.Increasing oil content and decreasing gossypol by transgenic and gene editing techniques can increase the supply of vegetable oil in China without occupying additional cultivated land and reducing fiber quality.Diacylglycerol acyltransferase(DGAT)is the last step of triglyceride synthesis and its expression level is closely related to oil content.Many previous studies have demonstrated that heterologous expression of DGAT can significantly increase oil content in seeds.In this study,four kinds of SiDGAT1,RcDGAT1,HaDGAT1 and AhDGAT1genes were cloned and transformed into Saccharomyces cerevisiae and cotton for identification,then creating cotton germplasm with high oil content.In this study,the gene editing technology was used to knockout the Gossypium PIGMENT GLAND FORMATION GENE(GoPGF),which laid a foundation for analysis of glandular formation regulation mechanism and the relationship between gland and gossypol.The main results are as follows:(1)Bioinformatics analysis and the yeast expression system showed that the cloned oil crops SiDGAT1,RcDGAT1,HaDGAT1 and AhDGAT1 all had DGAT1 characteristics and DGAT enzyme activity.(2)The SiDGAT1,R-DGAT1,HaDGAT1 and AhDGAT1 genes were overexpressed in cotton.Based on the results of southern blot,4,8,11,10 single-copy transformation events were screened,respectively.all of them were expressed normally in cotton;(3)Compared with the null plants,the SiDGAT1 transgenic T2 generation of Si-94,Si-99 oil content increased by 6.37%,7.41%,100-grain weight incre-ased b y 11.16%,4.94%,respectively;(4)Analysis of the lipidomics of SiDGAT1 transgenic cotton identified 8 types,26subclasses,902 lipid molecules and 25 differential lipid molecules were screened,including 14 TAGs.Triglycerides show an increasing trend with 56 or more carbon atoms and a quality of 900 or more(R~2=0.6771,R~2=0.6987).(5)Using CRISPR/Cas9 with two SgRNAs to knock out gossypium pigment gland formation gene GhPGF_A12 and GhPGF_D12.The 100%non-gland phenotype rate was observed.Compared with the control,the content of gossypol in leaves was decreased by98.8%,southern hybridization and gene editing proved that the non-gland phenotype was heritable.