Genotyping Of Two ZmZF Alleles And Its' Expression Study

The maize genome have much abundance of single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms(InDels), so it's very useful for developing molecular markers and discriminating alleles. At present, there are many procedures to assay SNPs and InDels. Allele-specific PCR is a simple, low-cost technique to discriminate the alleles quickly, but because of the mismatch between the specific primer and template made by the SNP site and 3'-end of primer, the result maybe not so accurate. For mazie allele discrimination, we can use the abundance of these polymorphisms when its be transformed to allele-specific PCR primers containing both of them, so this type of allele specific primer may reslove the problem of mismatched amplification made by single SNP or InDel site.In order to test whether this is a simple method to discriminate the alleles reliably and detect the alleles expression variation, the gene of ZmZF( Zea mays zinc finger-like protein) which is presumed to be a transcription factor gene induced by flooding stress and differently expressed in roots between two inbreds of Mo17 and Hz32 was chosen. In this study, we are want to establish a simple and accurate technique to discriminate alleles and detect the alleles expression variation in maize hybrid. The main results were:(1)Using a pair of primers designed according to the cDNA sequence published in the NCBI, we have amplified most of the gene's 3'UTR and small parts of the encoding sequence of the two alleles of ZmZF-Mo17 and ZmZF-Hz32. After sequencing,we have found three SNP sites and three InDel sites between the two alleles.(2)According to the AS-PCR primer design principle, we have designed several paris of genotyping primers which containing a SNP sites in the forward primer and InDel sites in the reverse primer. After tested, the allele specific primer of Pa,Ph(matched to the ZmZF-Hz32 for A type) and Pg,Pm(matched to the ZmZF-Mo17 for G type) were found. Further more, we have tested the stability of the genotyping primers in different Mg²⁺ and dNTP concentration. The results showed that the allele specific primers discriminated the alleles from parental inbred Mo17 and Hz32 specificly and perfectly. The allele specific primers also can be used in Real-Time fluorescence PCR method. (3)Using the semi-quantitative RT-PCR by the genotyping primers, the expression level of the alleles in the roots of the hybrid of F₁ and the parents were tested.The results showed that in the hybrid, the expression lever of the ZmZF-Hz32 allele has no obvious difference during the time of waterlogging ,but the allele of ZmZF-Mo17 was increased from the time on, and the expression lever of the parental allele of Hz32 was much more lower than the allele from the parent of Mo17. Using semi-quantitative RT-PCR to test the expression level of the leves also got the same results as the roots at Oh and 8h. Using the Real-Time fluorescence quantitative PCR method, we still found the expression variation in the hybrid and the two parental inbreds, but in the hybrid, at the 24h, the expression lever of the allele from the parent of Mo17 was lower than the 16h. These results suggested that the differential expression lever between alleles was common in the hybrid by flooding stress, and the allelic expression variation maybe caused by the difference of the alleles Cis factors.