| At present,the whole genome sequencing of rapeseed(Brassica napus)has been completed.Rapeseed has officially entered the post-genome era.Many mutant materials are needed to verify the function of genes.The construction of rapeseed mutant library can speed up the discovery of new mutants and the analysis of new gene functions.In this study,the pCB260-Kan plasmid was transformed into rapeseed by soaking method,and then the activation tagging insertion mutant library of rapeseed was established through Kan aqueous solution screening and PCR identification.A wax-deficient mutant(wad)was selected for detailed phenotypic analysis and transcriptome analysis.The main results of this study are as follows: 1.Establishment of mutant library and preliminary screening of mutant by KanPrevious herbicides were used in the laboratory to screen the transformed plants,resulting in the screened plants easily dying during transplantation.In this part,we replaced the herbicide resistance gene in pCB260 vector with Kan resistance gene,and transformed the pCB260-Kan plasmid on a large scale by soaking.Then we established a suitable screening method for rapeseed.Compared with spraying herbicides,Kan solution immersed seed screening is more convenient to control,and the selection pressure for each plant is more uniform.After preliminary screening with Kan solution,we obtained 8-9 seedlings per 500 seeds on average.The positive rate of Kan screening was between 1.6% and 1.8%.2.Identification of transformed plantsIn this part,we further identified the transformants screened by PCR,and found that most of the transformants screened by Kan were positive,which proved that the activation tag was inserted into rapeseed genome.We selected a wax-deficient mutant wad and tried to follow the reverse genetics strategy,using TAIL-PCR to obtain flanking sequences of the insertion tag.However,we tried a lot of TAIL-PCR and did not get the flanking sequences of the tag.3.Phenotypic identification of wadIn this part,we found that wad true leaf was shiny at the young stage but recovered at the mature stage.Toluidine blue staining and chlorophyll exudation experiments demonstrated that there was no obvious defect in the cuticle of wad leaf surface.However,it was confirmed by scanning electron microscopy that wad was indeed a wax-deficient mutant.We determined wax components in wad and found that wax with longer carbon chain than C24 decreased,which may be due to the blockage of fatty acid elongation pathway.For this reason,we determined the content of fatty acids in wad leaves,and found that there were defects in the absence of fatty acid elongation pathway in wad.The fatty acids greater than C24 decreased significantly,which proved that wad was a wax-deficient mutant caused by the obstruction of fatty acid elongation.4.Transcriptome analysis of wad mutantIn this part,we sequenced the transcriptomes of wad(wad-y)in young and wild type plants(WT-y),wad(wad-m)in mature and wild type plants(WT-m).Kyoto Encyclopedia of Genes and Genomes(KEGG)annotation showed that differentially expressed genes(DEGs)were gathered in the fatty acid elongation pathway.These genes were down-regulated in wad-y and returned to normal levels in wad-m,indicating that the changes in fatty acid elongation genes’ expression lead to dynamic changes in wax.Moreover,a transcription factor-enrichment analysis showed that BASIC PENTACYSTEINE 6(BPC6)is the key regulator affecting fatty acid elongation.Above all,we reported the specific wax-deficient mutant that shows a wax recovery phenotype at specific developmental stages.This mutant is important for the study of wax regulation in different developmental stages of Brassica napus.Moreover,we concluded that BPC6 is associated with the regulation of fatty acid elongation and affects wax biosynthesis.This work provides insights into the regulation of wax biosynthesis during different B.napus developmental stages. |
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