Creation Of GmWRI1a Gene Soybean Mutation Plants By CRISPR/Cas9

Triacylglycerols(TAGs)are major source of essential energy in the human life.Oil content and fatty acid composition are two important indicators to measure the quality of oil crops.Therefore,cultivating high oil content crop varieties is matter of concern to scientists.The use of traditional genetic breeding methods to achieve a substantial increase in soybean oil content exists objective obstacles.Therefore,it is one of the research methods to cultivate new high oil varieties by molecular biology which is to identify genes related to oil and fat synthesis,to analyze its molecular mechanism,and to improve the oil content of oilseed seeds by regulating the expression of these genes.Soybean is one of the most important oil crop.Improving its oil content and fatty acid composition is an important goals for soybean breeding.But,seed oil content of soybean cultivars is generally between 20-22%.And,high oil crops,such as peanuts,sesame,seed oil content is40-50%.Comparely,soybean seed oil content is lower.Thus,soybean has the potential to improve seed oil content.WRINKLED(WRI)is a member of the AP2 /EREBP transcription factor family.Current study shows that WRI1 plays an important role in controlling sucrose to oil accumulation and plays an important role in regulating of glycolytic and fatty acid synthesis pathways.In this study,the soybean Gm WRI1 a gene was as the research object.The soybean Gm WRI1 a mutant was created by CRISPR/Cas9,which depressed the expression of Gm WRI1 a in transgenic soybean.According to the results of the seed oil content of trangenic soybean and expresson level of genes involved fatty acid synthesis and glycolysis pathway,the role of Gm WRI1 a gene and regulation profile to fat biosynthesis were charactered.It is very important to further improve the quality of soybean by genetic engineering method.The main results obtained were listed bellow:(1)Three targets of soybean Gm WRI1 a gene were Gm WRI1a-sg RNA-1,Gm WRI1a-sg RNA-2 and Gm WRI1a-sg RNA-3.(2)Constructed Gm WRI1 a gene CRISPR/Cas9 binary expression vector pVK005-04-Gm WRI1asgRNA-1,p VK005-04-Gm WRI1 asg RNA-2 and pVK005-04-Gm WRI1asgRNA-3.The three plasmids were transformed into soybean by Agrobacterium rhizogenes.Target genome editings were tested by transgenic hairy root sequecing.The vector p VK005-04-Gm WRI1 asg RNA-1 and p VK005-04-Gm WRI1 asg RNA-3 were able to work.(3)The two plasmids were transformed into soybean by Agrobacterium tumefaciens-mediated cotyledon transformation.A total of 107 plants of T0 transgenic soybeanwere obtained 74 plants from Gm WRI1a-sg RNA-1 and 33 plants from Gm WRI1a-sg RNA-3.(4)Eight transgenic soybean Gm WRI1 a mutant were identified by herbicide resistance,PCR and DNA sequencing.The 8 transgenic soybean plants were numbered as follows: 310,327,287,294,350,282,109 and 250.(5)The expresson profile of oil synthesis-related genes of transgenic soybean Gm WRI1 a mutant was analyzed by Real-time PCR.The results showed that the genes related to oil biosynthesis were significantly down-regulated with the depressed expression of Gm WRI1 a in transgenic soybean GmWRI1 a mutant,which indicated that Gm WRI1 a gene was able to regulate the seed oil content of soybean by regulating glycolysis and fatty acid metabolism pathway.(6)The tests of T1 seed oil content of transgenic soybean Gm WRI1 a mutant showed that the oil content of transgenic lines were lower than that of wild type.The results indicated that the gene silencing of Gm WRI1 a was negnatively correlated with the total oil content of soybean seeds,and the Gm WRI1 a gene could regulate the accumulation of soybean oil.