Cloning And Characterization Of A Viviparous Mutant Gene And A Small Kernel Mutant Gene In Maize

Maize is one of the most important cereal crops, and its kernels have a wide range of uses.Therefore, it will be of great significant to explore the genes associated with maize kernel development.Kernel mutants are important resources for gene cloning and characterization, and some mutant genes can be directly used in maize production. Until now, researchers have cloned many maize genes with kernel mutants.Our laboratory had constructed a maize kernel mutant pool, using maize inbred line B73 and Mutator (Mu) active line. In this research, I will report the gene cloning and functional characterization of a maize viviparous mutant (vp-wl2) and a small kernel mutant (smk-wl10).The vp-wl2 mutant has white or pale yellow kernels, sprouting before harvest. The mutant endosperm accumulates zeta-carotene and embryos have dramatically reduced abscisic acid (ABA)content. The modified DLA method was used to clone the causal gene, and the results showed that a Mu9 insertion in the first intron of ZDS gene disrupted its normal transcription. Previous studies suggested that ZDS is likely the structural gene of the vp9 locus, or the transcription of ZDS gene may be regulated by the vp9 locus. In order to prove the relationship between vp9 and ZDS, an allelic test was performed. The results showed that vp-wl2 is a novel allele of the vp9 locus, so the ZDS gene is certified to be the structure gene of the vp9 locus. Transient expression of the ZDS-YFP protein in maize protoplasts suggested that the ZDS protein localized in chloroplasts. The ZDS protein is a key enzyme in the carotenoid biosynthesis pathway. Mutations in the ZDS gene will block the synthesis of carotenoids and reduce the content of ABA, and eventually lead to pre-harvest sprouting.The smk-wl10 is a small kernel mutant. The progeny of the heterozygote segregates normal kernels and small kernels in a 3:1 ratio. Some small kernels could germinate and produce stunted plants, which could develop normal tassels, while they failed to develop normal ears. The development of embryo and endosperm were delayed in most of the small kernels. The major morphological feature of BETL cell is wall-in-growth. However, the wall-in-growth feature could not be observed in mutant kernels, which will affect their capacity of nutrient transport. The map-based cloning approach was used to clone the causal gene and it was located to a 433 kb region on the short arm of chromosome 2. There are seven candidate genes within the interval, namely, ORF1, ORF2, ORF3, ORF4, ORF5, ORF6, and ORF7. The genomic DNA sequence of seven candidates are not changed in the smk-wl10 mutant, while the expression of ORF2 is substantially reduced in mutant kernels and seedlings. Further experiments certified that the CHG methylation level of ORF2 promoter region is significantly increased in the mutant. The ORF2 gene encodes a protein highly similar to the tubulin folding cofactor B protein.Moreover, mutation of the ORF2 ortholog gene in Arabidopsis leads to embryo lethal, so ORF2 is likely the causal gene of the smk-wl10 mutant.In conclusion, the results suggested that mutations in the ZDS gene will block the biosynthesis of carotenoids and reduce the content of ABA, which will lead to preharvest sprouting on immature ears.The mutation in maize tubulin folding cofactor B gene will reduce the normal development of maize kernel. These findings provided us with some novel evidences to understand the molecular mechanism of maize kernel development.