Molecular Cloning, Sequence Nad Expression Analyses Of Two Transcription Factors ZmC4HC3 And ZmNAC

Endosperm accounts for more than 80% of grain weight in maize. The development and filling of endosperm cell plays determined role in the formation of grain weight and quality. Three full-length cDNAs encoding different maize transcription factor were cloned in silico cloning and by RT-PCR approach using two ESTs derived from suppression subtractive hybridization cDNA libraries constructed in our laboratory. Predictions of the function domains and structurs of their proteins, their subcellular localizations and electronic mapping were also done by using bioinformatics algorithms.The main results were as follows:1. An EST (PE12C5) highly homologous to C4HC3 RING-type finger protein of rice was obtained from the SSH libraries during identification for differentially expressed genes between 10 DAP and 20DAP in popcorn inbred N04 endosperm. This EST was chosen as a query probe for in silico cloning. To verify the result of in silico cloning, specific primers were designed for RT-PCR amplification and a 1504bp cDNA fragment was obtained from 10 DAP endosperm. This fragment was fully sequenced and identified as a new maize C4HC3 RING-type finger protein (GenBank accession no. GQ131520.1). The full-length cDNA, termed ZmC4HC3, was consisted of a 237 bp 50-untranslated region (UTR), a 92 bp30UTR, and a 942 bp ORF which encoding for a putative protein of 314 amino acids. Comparison of the cDNA sequence with the nonredundant nucleotide database a predicted mRNA annotated as similar to C4HC3 RING-type finger protein was identified.2. Phylogenetic analysis of a multiple alignment showed that the C4HC3 RING-type finger protein had similarity to the C4HC3 RING-type finger protein from sorghum bicolor, rice and Arabidopsis, with similarities of 95%,85% and 70%, respectively. By performing a blast search against B73 RefGen_v1, ZmC4HC3 was located at bin 1.10.3. Another EST (DE57D10) highly homologous to NAC transcription factor of rice was obtained from the SSH libraries during identication for differentially expressed genes between 10 DAP and 20DAP in dent maize inbred Dan232 endosperm. This EST was chosen as a query probe for in silico cloning. To verify the result of in silico cloning, specific primers were designed for RT-PCR amplification. Two cDNA fragment, was obtained from maize endosperm 10 DAP,1267bp and 1359bp respectively. Both fragments were fully sequenced and identified as new maize NAC transcription factors. Their GenBank accession numbers were HM347322 and HM367095, and were named as ZmNAC and ZmNAC2. ZmNAC was consisted of a 79 bp 5'untranslated region (UTR), a 291 bp 3'UTR, and a 897 bp ORF which encodes a putative protein of 299 amino acids. ZmNAC2 consisted of a 79 bp 5'untranslated region (UTR), a 745 bp 3'UTR, and a 534bp ORF which encodes a putative protein of 178 amino acids.4. Phylogenetic analysis of a multiple alignment showed that ZmNAC was similar to the NAC transcription factors from rice and Triticum aestivum, with similarities of 86% and 80%, respectively. ZmNAC2 was similar to the NAC transcription factors from rice and Triticum aestivum, with similarities of 83% and 80%, respectively. Comparison of our two cDNA sequences with the nonredundant nucleotide database predicted mRNA annotated as similar to NAC transcription factor were all identified.5. The expression patterns of ZmC4HC3 in Dan232 and N04 were assayed by fluorescent quantitative RT-PCR. Befor 10d DAP, this gene showed higher expression in inbred Dan232 than in inbred N04, but it was an inverse between 10d and 20d DAP. A mong various tissues, it expressed simultaneously expressed in root, stem, leave, and embryo, with no tissue-specific expression. It expressed highest in 10d DAP in the leaf and the lowest both in the Dan232 and N04 endosperm, and highest in the stem and the lowest in the embryo in N04 20d DAP. It expressed highest in the pericarp and the lowest in the endosperm in Dan232 20d DAP.6. Bioinformatic analysis showed that ZmC4HC3 encoded a C4HC3 RING-type finger protein. GW2, a new QTL controlling rice grain width and weight, encoded a previously unknown RING-type protein with E3 ubiquitin ligase activity. The protein similarity of ZmNAC and ZmNAC2 with OsNAC6 was 59%. OsNAC6 was a member of the NAC transcription factor gene family in rice, which had been cloned and the function in gain weight had been proved. Therefore, three genes in this study, ZmC4HC3, ZmNAC and ZmNAC2 might play role in controlling gain weight in maize and could be studied in further researches.