大豆DGAT1/2基因表达分析和敲除靶点设计

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

二酰甘油酰基转移酶(DGAT)是将甘油二酯(DAG)催化成甘油三酯(TAG)的限速酶。TAG是植物种子油脂的主要储存形式,人体内TAG过高不仅会造成高血脂、高血糖,还会使肝脏功能超负荷代谢,导致肝脏功能受损,血清转氨酶含量增加并最终形成脂肪肝,而DAG则更有益于人体健康。为给创制大豆dgat突变体和解析DGAT1/2基因功能奠定研究基础,本研究利用定量PCR检测8个GmDGAT1/2基因(DGAT1a、DGAT1b、DGAT1c、DGAT2a、DGAT2b、DGAT2c、DGAT2d和DGAT2e)的表达模式,通过烟草叶片瞬时表达方法检测GmDGAT1c和GmDGAT2b表达对烟草叶片中油脂含量的影响,设计GmDGAT1/2基因敲除靶点并构建CRISPR/Cas9基因敲除载体,并通过大豆离体毛根转化试验检测基因敲除载体的有效性。结果表明:GmDGAT1/2在大豆的不同组织中均有表达,在种子中的表达丰度高于其他组织,且在完熟期(R8)的种子中表达丰度最高。瞬时表达GmDGAT1c和GmDGAT2b能使烟草叶片油脂含量分别提高32.53%和25.90%。另外,获得了含DGAT1a、DGAT1c的有效编辑靶点载体GmDGAT1-Cas9,含DGAT2a、DGAT2b、DGAT2c、DGAT2d和DGAT2e的有效编辑靶点载体GmDGAT2-Cas9。

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(DGAT1a,DGAT1b,DGAT1c,DGAT2a,DGAT2b,DGAT2c,DGAT2d and DGAT2e),expressed GmDGAT1c and GmDGAT2b 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 GmDGAT1c and GmDGAT2b separately was increased by 32.53%and 25.90%.In addition,we obtained vector GmDGAT1-Cas9 containing effective targets DGAT1a and DGAT1c,and vector GmDGAT2-Cas9 containing effective targets DGAT2a,DGAT2b,DGAT2c,DGAT2d and DGAT2e.