Research On The Nutritional Value Of Tomatoes Using Multi-gene Editing Technolog

Tomato is one of the largest vegetable crops produced and consumed in the world.In recent years,the breeding work of tomato is gradually extending to fruit shape,fruit color,fruit quality and so on,especially the fruit nutritional quality which is closely related to human health.Although studies on tomato nutritional value have been paid much attention,it is more challenging or difficult to cultivate varieties with multiple nutrient components through traditional crossbreeding and other methods with long cycle and low efficiency.The recently developed CRISPR/Cas9 gene editing technology can improve plant traits efficiently and accurately,providing a breakthrough tool for plant breeding.What’s more,CRISPR/Cas9 can edit multiple sites in the genome at the same time with great efficiency,opening up the possibility of creating tomato varieties that combine multiple nutrients.In this work,multiple gene editing techniques were used to simultaneously edit genes related to regulation of tomato nutrient metabolism,and to create a new tomato germplasm with multiple genes related to nutrient composition.The main results obtained are as follows:1.Exploration and establishment of efficient gene editing system in tomato.Tomato PDS gene(mutated albino)and mi R156 gene family were used to optimize the genetic transformation system and test the efficiency of single gene editing system and multi-gene editing system.The results showed that the editing efficiency of PDS sites was 100%.Four members of mi R156 were edited at the same time.The overall gene efficiency was 100%,and the single gene efficiency was 80%-100%.2.Selection and aggregation of nutrient-related genes in tomato.Multi-gene editing was performed on lycopene metabolism regulator SGR1 and Blc,gamma-aminobutyric acid metabolism regulator Sl GAD3,vitamin C metabolism regulator APX4,and steroid alkaloid synthesis pathway gene Sl7-DR2.Lycopene,γ-aminobutyric acid,vitamin C and vitamin D3 were increased synchronously in tomato to enhance the nutritional value of tomato.Two genes involved in sugar content,VP5 and Sl INVINH1,were also edited to improve the taste of the tomato.Multiplex gene editing vectors with different combinations of the above genes were constructed and tomato Micro-Tom was transformed.3.Acquisition of tomato nutrient-related gene editing materials.A series of multi-gene mutants related to multiple nutrient combinations were successfully obtained.For example,in SX-4-8 tomato editing lines,pure sum or double allelic mutations occurred in Sl7-DR2,Blc and APX4 genes,which is expected to increase the content of vitamin D3,lycopene and vitamin C.In SX-3-9 tomato editing lines,double allelic mutations occurred in Sl GAD3 and SGR1 genes,which predicted high accumulation of gamma-aminobutyric acid and lycopene.SX-5-4 tomato editing lines had double allelic mutations in Sl7-DR2 and Sl INVINH1 genes,which were expected to be rich in vitamin D3 and increased in sugar content.In this work,we established an efficient gene editing system in tomato,developed a series of efficient vector of nutrient-related genes,and obtained a variety of different nutrient-related gene aggregation materials,which provided a basis for the subsequent tomato quality breeding.