Development Of Insertional Mutant Library Using Tnt1 Retrotransposon In Brassica Napus

Oilseed rape(Brassica napus L.)is one of the four major oil crops in the world, which is crucially important for the world economy. With the accomplishment of B. napus sequencing, characterization of gene function is of great urgency. T-DNA and transposable elements are effective tools for generating mutant library which is very useful in functional genomics research. We tried to use the Tnt1 retrotransposon system to generate Brassica napus mutant library by using Agrobacterium mediated transformation. Since Tnt1 retrotransposon element will randomly jump among the chromosomes under stressed condition, we chose the positive lines from T1 generation to create new mutant lines by tissue culture. After that, we tried to get flanking sequences by TAIL-PCR and adapter-PCR.We also attempted to hybridize part of transgenic plants with Brassica rapa(yellow sarson). The research contents and results are as follows:1. We obtained a total of 201 transgenic plants, and the positive rate was 56%.2. Plant tissue culture used T0 transgenic seeds numbered T0-7 and T0-15.We obtained 103 T1z-7 transgenic plants, the positive rate was 88%.We obtained 135 T1z-15 transgenic plants, the positive rate was 97%.3. For isolating the flanking sequences of T0 generation, TAIL-PCR and adapter-PCR were conducted using three nested specific primers designed based on the sequence of the vector Tnk23.Sequence analysis showed that 34 of the total 57 were B. napus genomic DNA, 13 were non-repetitive flanking sequences.We also verificated the Tnt1 insertion site, and the expression of the gene was researched later.4. We focused on leaf 、flower and plant traits, recorded the phenotype of T1 transgenic plants in the spring of 2016.5. We transmitted the Tnt1 fragment into Brassica rapa by distant hybridization. Transgenic plants numbered T1-15, T1-33 and T1-96 were hybridized with Brassica rapa and seeds were harvested in May of 2016.