Cloning And Characterization Of Profilin3 Gene And Its Promoter Region From Maize (Zea Mays L.)

Profilin is an actin monomer binding protein with a predicted molecular weight of 12-15kD, which exists in all of the eukaryote cells. Depending on the different conditions, profilin gene family causes both polymerization and depolymerization of actin filaments. It's presumed that profilin gene may play a significant role in signal transduction and regulation in the actin reconstruction during pollen germination and pollen tube elongation. Recent researches reveal that profilin also take part in RNA transcription and regulation of pre-mRNA splicing. In a word, profilin gene have comprehensive significance on physiology.According to maize S-Mo17^Rf3Rf3 cDNA microarray data, a profilin3 gene was found upregulated in the later stage of pollen development.In this article, cloning of the profilin gene and its promoter region, and function analysis of it were carried out. Primary results are listed as follows:1,Full-length cDNA of this profilin gene, named ZmPRO3 was cloned (GenBank accession number: EF546785) in maize S-Mo17^Rf3Rf3 pollen using RACE. cDNA sequencing indicated that the full length of ZmPRO3 cDNA is 864 bp, which contains a 396 bp open reading frame encoding 131 amino acids. Analysis shows ZmPRO3 protein has a profilin conserved domain,and its amino acid sequence is highly homologous to sequences of other plant profilins. Furthermore, characterizations and structures of ZmPRO3 protein were partially predicted, too.2,The whole ORF and the corresponding genomic DNA of one pair of Near Isogenic Lines (NIL), S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3 were respectly amplified using PCR, and the ordinal sequence length was 396bp, 585bp and 570bp. The structure of ZmPRO3 from the two of the NIL was predicted using GENSCAN tools. The prediction results showed that their genomic DNA both comprises 3 exons with 2 introns conservatively inserted in their coding regions, but the corresponding length of introns and the splicing sites of their second intron were different from each other. By using Clustal W tools, the genomic sequences of S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3 were aligned,and the results indicated that there exists 48 (8.2%)mutant sites, of which 23(3.9%) sites are deletion mutations, and the left sites are spot mutations.By far, the analysis of these genomic differences is undergoing. 3,One pair of Near Isogenic Lines (NIL), S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3 were used to characterize gene expression analysis of ZmPRO3. Northern blot results indicated that ZmPRO3 does not express in non-pollen tissues, but the expression of ZmPRO3 differed significantly in pollen between S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3. That is to say ZmPRO3 does not express in pollen of S-Mo17^rf3rf3, but shows opposite expression level in pollen of S-Mo17^Rf3Rf3. This result is consistent with the hybridization result of Zhang's microarray. Meanwhile, the expression differences suggested that ZmPRO3 may take part in the normal pollen development processes.4,By using chromosome walking technique, promoter regions of ZmPRO3 gene were cloned from S-Mo17^rf3rf3 and S-Mo17^Rf3Rf3. Sequencing results indicated that the length of promoter region we cloned was 604bp, and it's totally indentical between S-Mo17^rf3rf3 and S-Mo17^rf3rf3. According to alignment results of the promoter sequence, it comes to be a new one first cloned, and now has been registered in GenBank with the accession number EF565370. Four TATA box,five CAAT box,seven GC box,several cis elements which have significance for initiating gene pollen-specific expression, and a series of cis elements relevant to stresses were found in the promoter region. So, we presumed that promoters we cloned was likely to have pollen-specific regulation on ZmPRO3 gene expression, and ZmPRO3 may response to several biotic and abiotic stresses, but they all need to be proved.