The Construction Of TT8Interference Vector And Genetic Transformation Of Brassica Juncea (L.)

Oilseed rape is one of the four major oil crops in the world, its seed coat color is not only a morphology indicating traits and related to the seed dormancy, but also related to the quality of oilseed rape[1].In order to establish an efficient genetic transformation system for Brassica juncea (L.), we used hypocotyls of Sichuan yellow seed and Brown mustard of Brassica juncea (L.) as explants with Agrobacterium-mediated transformation, which optimized in three aspects from the callus induction, bud differentiation and root culture with different combinations of hormone and silver nitrate. At the same time, we used TT8as the research object and constructed a inference vector with TT8. To analysis the function of TT8gene, we transformed NILA of Brassica juncea (L.), which can lay the foundation for breeding high oil and quality yellow seed oilseed rape varieties. The results are as follows:1. Optimized the genetic transformation system of Brassica juncea (L.), which used the hypocotyls as explant with Agrobacterium-mediated. On the basis of basic culture medium, by adding1.0mg/L of2,4-D to pre-culture,3.0mg/L6-BA and5.0mg/L AgNO3to shoot differentiation,2.5mg/L IBA and0.05mg/L NAA to root. The hypocotyls callus regeneration rate, shoot regeneration rate and root rate were100%,28.33%and100%respectively. The positive rate achieved23.80%with PCR and GUS staining detection, which illustrated that the system was feasible.2. We integrated the TT8to pfGC5941vector through the molecular cloning technology such as enzyme digestion and connection, and constructed the interference vector pRNAi-TT8successfully.3. We obtained15positive plants through transforming NIL A of Brassica juncea (L.), and10of them gained seeds successfully. The rate of positive transformation was38.46%. Compared to CK seeds, the color of To plants was lighter and its seed plumpness decreased, which indicated TT8genes played a role in the seed coat proanthocyanidins synthesis of Brassica juncea (L.). But it remains to be further studied its participation mechanism. Detected the T1plants young leaves’DNA by PCR, our results showed that the exogenous gene TT8separated in varying degrees and the growth of transgenic offspring was stable and the exogenous gene can be stably inherited. The T1plants are podding, we have to wait the T2generation seeds to be harvested and then identify the phenotypes and analysis the content of its related compounds.