Phenotypic Analysis And Gene Mapping Of Maize Defective Kernel2014 (dek2014) Mutant

It is important to investigate the molecular mechanism of kernel development because that kernels have a big impact on maize yield. A natural kernel mutant defective kernel2014(dek2014) was used as the experimental material in this study. The immature kernels of mutant showed light color, enlarged size. There are a large number of liquid substances in it. The mature kernels of mutant have shrunken phenotype. The endosperm were severely reduced and embryo was lethal. Genetic analysis showed that the mutant phenotype is controlled by a single recessive locus. The candidate locus were narrowed down to an 113-Kb region between molecular marker 10-49(Bin:3.09) and 10-54(Bin:3.09) at the end of the chromosome 3 long arm by map-based cloning technique. There are three candidate genes in the region. Among them, GRMZM2G579005 is transposable element that does not encode any protein, and GRMZM5G842494 encodes protein of unknown function. For the third candidate gene, GRMZM2G429899, known as gene SHRUNKEN2(SH2), encodes the large subunit of key rate-limiting enzyme AGPase that plays an important role in starch biosynthesis pathway. The mutant locus is located in about 4200-4700 bp interval upstream of SH2 based on the result of sequencing. It contains two deletions(Del1 and Del2) and two insertions(Ins1 and Ins2). The candidate gene GRMZM5G842494 locates between the mutant locus and gene SH2, only 1047 bp upstream of SH2. The result of qRT-PCR indicated that SH2 was expressed specifically in wild-type endosperm and GRMZM5G842494 was mainly expressed in wild-type embryo. However, the expressions of both genes were significantly reduced in the mutant kernels. Further, the effects of Del1, Del2, Ins1 and Ins2 on gene transcription were analysized using transient expression system of tobacco leaves. The results showed that Ins2 had the most influence on transcription of downstream genes, therefore, it may be the main factor leads to abnormal expression of two candidate genes.We crossed the sh2 mutant with heterozygous dek2014 mutant. The 1/3 progeny kernels have abnormal endosperm but normal embryo. So, we proposed that the mutant phenotype of dek2014 mutant may controlled by both GRMZM5G842494 and SH2. SH2 controlls the endosperm development. GRMZM5G842494 regulates the embryo development, so we named GRMZM5G842494 as DEFECTIVE EMBRYO1(DE1). To further investigate the function of DE1, we constructed both overexpression vector and gene knockout vector of gene DE1, and try to determine the gene function in the regulation of embryonic development.Taken together, the Ins2 in dek2014 mutant resulted in the severely reduced transcription of downstream genes DE1 and SH2, which affects the kernel development. Of cause, the more molecular mechanisms were remained to be investigated.