The Establishment And Optimization Of Tagetes Erecta VIGS System

Tagete erecta, belonging to the Tagete genus of the Asteraceae family, has great important ornamental and economic value. As an emerging technology, Virus-induced gene silencing plays an important role in the study of gene function in plants. Our subject has established a comprehensive system of Tagete erecta VIGS, which laid the theoretical and technical basis for building Virus-induced silencing system in other compositae plants.The main research results were showed as the following several aspects:(1) Construction of Tagete erecta VIGS vector on the basis of Tobacco Rattle Virus(TRV) vector. We have successfully cloned a 417 bp size of report gene PDS from Tagete erecta, and constructed the recombinant viral VIGS blank vector and vector absence and presence of Te PDS segment.(2) Establishment and optimization of Tagete erecta VIGS system at the stage of cotyledon. We chosen Tagete erecta plants at the stage of cotyledon as this experimental material and observed Tagete erecta plants with the report gene PDS silencing by vacuum infiltration method. The desired result of report gene PDS silencing in Tagete erecta plants based on the cotyledon stage were obtained by vacuum infiltration for five minutes under the background of the bacterial concentration OD600 values being set to 0.5, 1.0, 1.5and 2.0, osmotic strong being set to-25 kpa. Comprehensive evaluation of the impact of osmotic pressure(-25 kpa,-50 kpa,-100kpa) and length of treatment time(2min, 5min,10min) on the experiment results, our study concluded that the optimal penetration condition is basing on the intensity of the osmotic pressure-25 kpa, penetration time of 10 minutes basis.(3) The research of VIGS translation in Tagete erecta leaves. We took oppression injection with bacterial concentration OD600 = 0, 1.0, 2.0 and 4.0 to infect the fourth pair of true leaves of Tagete erecta plants. The study results showed that Tagete erecta plants leaves in the VIGS::Te PDS experiments and the control experiments(VIGS::blank)appeared slight yellow phenomenon rather than photobleaching.(4) The research of VIGS translation in Tagete erecta buds. We introduced steminjection、root-irrigation and vacuum infiltration into our expertment to treat the fourth pair of true leaves of Tagete erecta plants, and then observed the Tagete erecta plant leaves and buds phenotype. The results showed that the Tagete erecta plant leaves and buds with treating by root-irrigation did not display any phenotype, on the contrary, the Tagete erecta plant leaves with treating by stem injection and vacuum infiltration appeared photobleaching, but under the same conditions the Tagete erecta plant buds showed without any change. Taking stem injection to infect the second、third、fourth、fifth and sixth pair of true leaves of Tagete erecta plants, Over a period of time we observed the leaves photobleaching at the top treatment site, but buds having nothing phenotypic changes. Tagete erecta plant leaves appeared photobleaching phenotype by injecting once or twice to Tagete erecta plant buds(including torus and peduncle), while only 15% in the case of continuous treatment of Tagete erecta plant buds, we could find sepal turning into photobleaching phenotype.