Gene Target Design and Functional Analysis of Soybean DGAT1/2 Genes

Diacylglyceryl transferase（DGAT） is a speed-limiting enzyme that catalyzes triacylglycerol（TAG） from diacylglycerol（DAG）. TAG is the main storage form of plant seed oil. Excessive TAG in human will only not cause hyperlipidemia and hyperglycemia, but also overload metabolism of liver function, resulting in impaired liver function, increased serum transaminase content and eventually formed fatty liver. While, DAG is more beneficial to human health. In order to lay the groundwork of acquire the mutant and understanding the function of DGAT1/2 genes, using quantitative PCR to detect tissue expression patterns of the eight GmDGAT1/2 genes（DGAT1 a,DGAT1 b,DGAT1 c,DGAT2 a,DGAT2 b,DGAT2 c,DGAT2 d and DGAT2 e）, expressed GmDGAT1 c and GmDGAT2 b separately in tobacco leaves. And we constructed the knockout vectors of DGAT1/2 and transformed them into soybean hair roots for target detection by CRISPR/Cas9 technology. The results showed that GmDGAT1/2 were expressed in all detected tissues of soybeans, and the abundance of expression in seeds was higher than that in other tissues. And the expression of them in the seeds was the highest at seeds of mature stage（R8）. The fat content of tobacco leaves, which expressed GmDGAT1 c and GmDGAT2 b separately was increased by 32.53% and 25.90%. In addition, we obtained vector GmDGAT1-Cas9 containing effective targets DGAT1 a and DGAT1 c, and vector GmDGAT2-Cas9 containing effective targets DGAT2a, DGAT2b, DGAT2c, DGAT2d and DGAT2e.