Research Of Obtain High Lycopene Engineered Tomato Plants By Transferring MiR394

Micro RNA(miRNA)is a type of short(about 21 nucleotides)endogenous non-coding RNA that can regulate gene expression in eukaryotes.miRNA is almost involved in all biological processes of tomato,especially development,differentiation,Maturation,senescence,and biotic and abiotic stress responses.Mi R394 is an ancient and well-preserved miRNA in plants.It is involved in the regulation of plant development and stress response.It exists in many dicotyledonous and monocotyledonous plants,such as Solanum lycopersicum,Arabidopsis thalian,Brassica napus,Citrus sinensis,Medicago truncatula,Solanum tuberosum,Vitis vinifera,Zea mays,Oryza sativa.At present,the role of miR394 has been explored in Arabidopsis,rice,rape and other species,but there is no research report on miR394 in tomato.In this study,based on the optimized and established tomato regeneration system and genetic transformation system,the constructed p FGC5941-miR394 overexpression vector was transferred into Micro-Tom tomato through the leaf disc method mediated by Agrobacterium,and high tomato red was obtained.Vegetarian quality transgenic plants.The main findings are as follows:1.Optimized a simple and stable Micro-Tom tomato regeneration system.To study the effects of different disinfection methods on the germination rate of seeds,and the effects of different hormone ratios on the induction of callus,adventitious buds and roots,and establish a stable tissue culture regeneration system.Finally,the optimal regeneration system is:disinfected with 5% sodium hypochlorite for 20 minutes,the germination rate of Micro-Tom tomato seeds is higher;the most suitable for inducing callus and adventitious buds is: 2.0mg/L zeatin(ZT)combination 0.1 mg/L indole acetic acid(IAA);0.5 mg/L indole butyric acid(IBA)is most suitable for inducing root regeneration.2.The Micro-Tom tomato genetic transformation system was established.The factors affecting the efficiency of genetic transformation were explored: the infection concentration of different Agrobacterium liquids,the infection time,and the concentration of bacteriostatic antibiotics.Through comparative analysis,it was found that the infection concentration of Agrobacterium solution was OD600=0.4,the infection time was 5 min,and the growth and differentiation of explants was the best.The concentration of the bacteriostatic antibiotic Timentin was 300 mg/L.The bacteria have the best effect and the conversion rate is higher.3.Using the optimized and established system,the p FGC5941-miR394 overexpression vector was transformed into the wild-type Micro-Tom tomato,and the positive strain was obtained after identification.Through real-time fluorescence quantitative q RT-PCR analysis,the expression level of miR394 gene in the transgenic plants was 4 times higher than that of the wild type.It was observed that the tomato fruits of the transgenic plants in the same period were darker and redder than the wild type.The HPLC results showed that in the three independent transgenic lines,the lycopene content of the tomato red ripe fruit was increased by about 74.16%,83.55% and 63.57%,respectively,compared with the wild type.Compared with WT,the expression of lycopene biosynthesis genes ZDS,PDS and PSY1 showed a rich accumulation,which was significantly up-regulated by 12.9-13.3,12.6-13.0,12.9-13.7 times compared with the wild type.