Construction Of Mutant Library With Activation-tagging Approach In Brassica Napus

Brassica napus L. is one of the most important oil crops. The accomplishment of the B. napus whole genome sequencing makes the research of post functional genomics more and more important. While mutant is an important basis for researches on gene function, it is effective to create gain-of-function mutant with activation-tagging which possesses special advantages on elucidating unknown genes or the unknown function of redundant genes. In this study, an activation-tagging mutant library of B. napus was constructed using agrobacterium-mediated transformation. The library was then preliminarily studied with analyzing its insert-shear model of T-DNA tagging, genome-wide distribution of T-DNA insertions and gene expression analyses at flanking regions. The results were summarized as following:1. The genome of Westar, a wild type of B. napus, was treated as a recipient of activation-tagging. Based on this, an activation-tagging mutant library was then constructed consisting of 285 positive independently transformed lineages which were identified with marker gene Npt II.2. The phenotypic traits were investigated in field for 2 consecutive years. Comparing to the wild type, transgenic lineages showed diverse observable variation on phenotypic characters including leaf shape, effective branches number, pollen fertility and flower morphology et al.3. Using thermal asymmetric interlaced PCR and adaptor ligation-mediated PCR, 27 flanking sequences were isolated and further mapped to B. napus reference genome. Three types of sequence shearing were found at the left boundary of T-DNA: lost partial nucleotides(< 25bp) but reserved a definite boundary; lost most nucleotides(> 50bp); and lost the entire boundary sequences. Furthermore, it indicated that no significant preference was observed between T-DNA insertion sites on B. napus subgenomes A and C.4. RT-PCR was used to detect the expression quantity of candidate genes in the enhancer flanking regions. Relative quantitative analysis of 2-ΔΔCT indicated that expression level of the 4 candidate genes showed no significant difference between the mutant and wild type.