The Cloning And Preliminary Function Validation Of Zmll-dap-at In Maize

Endosperm accounts for more than80%-85%of grain weight in maize. Thedevelopment and filling of endosperm cell plays determined role in the formation of grain weightand quality. Two full-length cDNA sequences of Zmll-dap-at were cloned from two inbreds ofdifferent grain size. The seed sequence EST predicted as transaminase was selected from thesuppression subtractive hybridization cDNA libraries constructed in our laboratory. We forecastedthe function of the gene，and analysed the relative expression volumes (REVs) of the two inbredlines in different tissues and development stages. The Prokaryotic expression and genomesequence analysis were also done in our study.The main results were as follows:1. An EST (DM18D5) was obtained from the SSH libraries during identification fordifferentially expressed genes between10DAP and20DAP in popcorn inbred N04endosperm.This EST was chosen as a query probe for in silico cloning. To verify the result of in silicocloning， specific primers were designed for RT-PCR amplification and a1763bp cDNA fragmentwas obtained from20DAP endosperm. This fragment was fully sequenced and identified as amaize aminotransferase (GenBank accession no. GU180091.1). The full-length cDNA， wasconsisted of a129bp5’-untranslated region (UTR), a230bp3’-UTR， and a1386bp ORF whichencoding for a putative protein of462amino acids. Comparison of the cDNA sequence with thenonredundant nucleotide database， the gene was belonged the aspartate aminotransferase(AAT)-like subtribe of the AAT-I superfamily.2. Phylogenetic analysis of a multiple alignment showed that the Zmll-dap-at had91%and76%identities with that of the DAP-AT sequences from rice and Arabidopsis， respectively. Buthave lower homology to the bacteria， the percent were below65%.By performing a blast searchagainst B73RefGen_v1， Zmll-dap-at was presumably most likely to be located in corn first, fourand nine on chromosome.3. Real-time PCR (RT-PCR) analysis showed that the expression of Zmll-dap-at was nottissue-specific， but was various between two inbreds， among different tissues and graindevelopmental stages. Relative expression volumes (REVs) were consistently higher for Dan232 than N04in all cases. For the two stages， REVs were higher at20DAP than at10DAP for alltissues except the opposite in leaf.4. Using the vector pET-28constructed the recombinant and induced the protein expression.SDS-PAGE electrophoresis results showed that the expression quantity of protein in size43kDawas increased obviously.5.15maize inbred lines of different lysine content were selected, and the genome sequenceswere amplification. Analysis of the promoter regions, we divieded the inbred lines into twoclass.The main difference between the two class is a region of11bp. inbred lines of lysine contentis higher.In this region, Lys content of inbreds including the11bp nucleotides were higher.