Cloning Full-length Of ZM401, A Non-coding RNA Gene From Zea Mays, And Functional Characterization In Pollen Development

The recent completion of the sequence of the Arabidopsis genome and the human genome have led to the identification of thousands of new putative genes based on the predicted proteins they encode. Genes encoding tRNAs , ribosomal RNAs, and small nucleolar RNA has also been annotated; however, a potentially important class of genes has largely escaped previous annotation efforts. These genes correspond to RNAs that lack significant open reading frames and encode RNA as their final product. Accumulating evidence indicates that such "non-coding RNAs"(ncRNAs) can play critical roles in a wide range of cellular processes, including chromosomal silencing, transcriptional. regulation, developmental control, and responses to stress.In our previous study, A cDNA library to poly (A) RNA isolated from mature pollen of zea mays was constructed. Differential screening and Cold-plaque screening obtained one cDNA fragment; designated ZM401 (Zea mays), was specifically or preferentially expressed in mature pollen. According to the ZM401 cDNA fragment sequence, full length of ZM401 cDNA was generated by 5' and 3' RACE method. ZM401 cDNA was 1149bp in length. Within the full-length cDNA sequence, a clear open reading frame (ORF) was undectable by omega 2.0 and DNAMAN software. The longest potential ORF was 269 nucleotides long (791-1060), coding 89 AA. But it had a poly (A) tail. All these results suggest that ZM401 was one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as RNA. RT-PCR and Northern blot analyses showed the ZM401 was transcribed from tetrad stage of microspore development and increased in concentration up to mature pollen; that suggested ZM401 belongs to late gene in pollen development. Two transcripts of the ZM401 gene were detected by northern blot analysis (1.2Kb, 2.0Kb). Southern hybridisations showed that the ZM401 in corn was present in one or a very few copes in the genome.To gain more information about the function of ZM401 gene, plant expression vectors with sense and antsense ZM401gsne promoted by 35S promoter or ZM13 promoter were constructed. Maize embryogenic callus were transformed by microprojectile bombardment with four plant expression vectors. After selecting for hygromycin resistance, 476 regenerated plants were obtained. 13 plants with sense ZM401 and 14 plants with antisense ZM401, respectively, were obtained by genomic PCR, dot bloting, southern blotting analysis. The aberrant phenotypes were observed in the anther of the transgenic plants with sense ZM401 gene. Most of the pollen grains in the transgenic plants with sense ZM401 were distored and were not stained with the solution of VIK, indicating the absence of accumulated starch. In contrast, most of the pollen grains in the non-transformant were rounded and were deeply stained by solution of I2-IK. The pollen viability of 13 independent transgenic plants with sense ZM401 was reduced between 1.2% to 18.2%, while that of non-transformants was more than 90%. Histological differences were observable for transgenic plants with sense ZM401. Comparing the non-transformant with transgenic plant, microspores in the non-transforming were rounded normally; the shape of most of the pollen grains of transgenic plants became distorted. The degradation of thetapetum was delayed for transgenic plants. The synchronism of pollen development and pollen sac fusion were destroyed. All these results showed that ZM401 gene play an important role during pollen development.