Cloning The Mu Element Insertional Mutant Gene Mio16 Of Maize Using Double Selected Amplification Of Insertion Flanking Fragments (DSAIFF)

Maize genome contains a large number of transposon and retrotransposon sequences. All various actived maize transposon are useful to construct inserted mutant pool. Maize Mutator (Mu) transposon has become an effective tool for creating maize mutant and conducting molecular biology study in virtue of its characteristic that induced high-frequency forward insertion mutation.In this study, an opaque endosperm [named Mu-inserted opaquel6 (mio16)] was chosen as test material from the Mu-inserted mutant pool provided by Dr. Yang Wenpeng. The opaque endosperm was used as a phenotypic marker to screen the mio16 mutation, the mio16 was backcrossed with the recurrent parent QCL2179, a wild-type background with vitreous endosperm, to produce a BC3F2:3 family; a modified method namely Double Selected Amplification of Insertion Flanking Fragments (DSAIFF) was employed to identify the Mu Flanking Fragment (MFF) of mio16;the co-segregation analysis was employed to verify linkage between the target gene and phenotype in BC3F2:3 family; basing on published genomic sequences of maize, the silico mapping of mio16 was done with bioinformatics study methods;meanwhile, according to the gene sequence, the primers were designed to detect the RNA expression of mio16; the full-length cDNA of the wild-type was obtained by RT-PCR primer-scanning technique and analyzing the Mu insertion site. This study gained these following conclusions:1.DSAIFF was effectively applied to isolate ambi-MFFs of mio16:Msel-MFF and MspI-MFF.2.The same 9-bp TSDs were found to exist in the Mspl and Msel digested MFFs, these MFFs were verified to be the two flanking sequences of an identically inserted gene.3.Co-segregation analysis suggested that these two MFFs were associated with the mutant opaque endosperm. 4. The result of silico mapping suggested that mio16 was mapped into the maize chromosome 4.09 bin.5.Mio16 gene contains a 2550bp open reading frame (ORF) encoding a putative 850aa protein and is composed of 11 exons and 10 introns.Mu insertion site is the+35 downstream of the TSS in the 5'-untranscription region (UTR).6.The RT-PCR result of mio16 RNA expression showed that the expression of miol6 has been changed because of Mu element insertion, and mio16 may be multi-copies.