| Maize germplasm is the base of maize improvement. The genes for breeding focus on a small part of germplasm with the improvement of production levels and popularization of new cultivars, which leads to a serious loss of genetic diversity. Now, the centralized use of excellent germplasm and the narrower germplasm resource have been the dominated restriction factors for current and future corn breeding. The mutations induced by physical and chemical factors will create brand-new genetic types and enrich maize germplasm. But, traditional mutation breeding have recently no great advances with the problems of lower mutagenic efficiency, more nonsense mutation and select difficult of beneficial mutations.Targeting induced iocal lesions in genomes (TILLING) provides maize reverse genetics research a new method. TILLING will effectively combine chemical mutagenesis (high frequency point mutations), directional PCR screening technology with two-color infrared fluorescence detection technology. It uses ethylmethane sulfonate (EMS) to induce a series of point mutation before doing RT-PCR amplification and high resolution melting (HRM) analysis with designed specific primers, which will quickly and efficiently identify point mutations from the mutations group, whose characters include plant structure, organic shapes, growth and development, yield and quality components and pest and disease resistances and all of which have brought new germplasm for maize breeding improvement. This research has used EMS pollen mutagenesis technology to construct mutant library. Then, we chose useful mutants by observing the field properties of mutagenesis progeny. Meanwhile, in Mi generation, we did gene analysis of opaque (the transcription factor gene which affects the quality of corn endosperm protein) and TILLING analysis of CENH3(proteins specifically histone3gene which affects the mitosis process of centromere) to provide new germplasm for high quality protein maize breeding and new haploid induction system.We did preliminary experiment of choosing two model of inbred lines (B73, Mol17) and two southwest biome backbone inbred lines (18-599R,18-599W) to be tested materials, whose pollen were disposed by EMS-paroline treated solutions for45minutes by different EMS volume fractions (0(CK),0.50×10-3、0.67×10-3、1.00×10-3、1.67×10-3 2.00×10-3) respectively. The seeds were harvested to calculate setting percentage and determine the appropriate EMS induced concentration.By the result of preliminary experiment, this research adopted the1.0×10-3volume fraction EMS-paroline treated solution to deal with18-599R pollen and the field observation showed that M1generation had a lower rate of emergence, plant formation and larger variation of seedling appearance compared with control groups. The changes at seedling stage had referred to color and shape variations of leaves. In addition, M2generation had a poor performances compared with control group and appeared large variations of grain phenotypes, even a beneficial mutation of sweet corn.This research extracted the genome DNA of mutagenic M1generation to construct8-fold DNA pools. According to the gene opaque and CENH3exon sequence, we designed13groups of specific primers to do Real-Time PCR amplification in DNA pools and HRM analysis. At last, we found8mutated materials.After the confirmation of sequencing, the8mutated materials had3base mutations compared with control sample, two of which lied in intron area causing no influence for gene expression. No.3134nucleotide in the fourth exon of opaque gene changed from G：C to A: T, which resulted in the alternation of coded amino acid form arginine to histidine. By the analysis of bioinformatics, this mutation located in bZIP domain of02protein, which composed the bipartite NLS B (between223and235amino acid site) in the Alkaline domain of02. As the chief factor of02core target, this mutation of amino acid may influence the efficiency of02protein transportation to the nucleus. Therefore, it is likely to change the process of transcription factor into the nucleus as well as the activity and combination of transcription factors for DNA. Therefore, it may affect the specific expression of transcription factor opaque in the endosperm, which needs further evaluation of its impact on endosperm protein quality. |
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