The Construction And Evaluation Of Small Size Inducible Expression Mutant Library Of Brassica Napus Seed Gene In Arabidopsis

FOX-hunting mutant library provided an important platform for the research of Arabidopsis and rice functional genomics.Compared with model plants,the platform of Brassica napus functional genomics is relatively backward,especially in the aspect of brassica napus mutant library.In this research,the seeds of different development stage were used as the first material,using GATEWAY recombination technology we constructed a cDNA library,then based on the cDNA library we constructed an inducible expression library for transforming Arabidopsis,at last we got a mutant library which over express the transgenes when being induced.Through evaluation,preliminarily we consider the mutant library could be used for the research of brassica napus functional genomics.The results are listed as follows:1.Through the analysis of Brassica napus cDNA library,we found that the full-length and non-redundant cDNA percentage is 43.45%.2.We constructed a inducible expression plant binary expression vector of Brassica napus genes,through transforming Arabidopsis,we have gained 3,200 T1 transgenic lines and harvesting their seeds independently.3.We selected 226 T1 transgenic lines,isolated the cDNA by PCR and sequenced the cDNA.We found that the full-length and non-redundant cDNA percentage is 36.58%.4.After sowing the seeds of 2,399 T2 transgenic lines,inducing the cDNA expression and screening the phenotype of morphology.We have found 25 visible mutants that caused by cDNA over-expression,they are 2 leaf narrow lines,19 early senescence lines,2 dwarf lines and 2 lines with abnormal siliques.5.Harvesting the selfing seeds(T3 generation)of 2,399 transgenic lines,After detecting oil contents of the seeds,we found that the oil contents range from 25%to 40%.Among them the oil contents of 4 lines were up to 40%.This result is been confirming.6.We have isolated the Brassica napus cDNA of the 25 mutants and got their sequences.In brief,using Arabidopsis as ectopic host constructing inducible gain-of-function mutant library can be used for the research of Brassica napus functional genomics.The mutant library is a valuable material resource.