The Expression And Functional Characterization Of MiR158 During Pollen Development In Brassica Campestris Ssp. Chinensis

Plant microRNA (miRNA) is a kind of plant endogenous non-coding RNA, which is involved in many plant growth and development processes through negatively regulating the expression of its target genes. Pollen development is an important part in the sexual reproduction of seed plants, in which many miRNAs have been reported to be involved. In our previous studies, we have conducted a high throughput sequencing for the pollen development related miRNAs in Chinese cabbage (Brassica campestris ssp. Chinensis) ’Bcajh97-01A/B’. We noted that miR158 had differential expression patterns between the male sterile line and the fertile line, and we also found that over-expression of miR158 could cause pollen aberration.In this research, we continued to study the function of miR158 in the pollen development process. We first cloned the two precursor genes and the target gene of miR158, and then we analyzed the temporal and spatial expression patterns of the two precursor genes by qRT-PCR and promoter-GUS reporting system. Finally, we explored the function and possible working patterns of miR158 and its target gene in Chinese cabbage by observing the phenotypic changes of over-expression or inhibition of miR158. The main results and conclusions are as follows:(1) The two precursor genes of miR158 were cloned according to their hairpin sequences. These two genes are located on chromosome 5 and chromosome 8, respectively, and its transcription products can form stable secondary structures, which are highly conserved in the miRNA and miRNA* regions, this is consistent with the evolutionary characteristics of miRNA, and also implies the conservation of the function of miR158. Meanwhile, synteny analysis showed that the two precursor genes were located in genomic regions corresponding to the region of miR158a in Arabidopsis, which means they may be retained from Ath-miR158a after genome triplication in Brassica. Therefore, we named these two genes as Bra-miR158al and Bra-miR158a2.(2) We also cloned the DNA and ORF sequences of BraO27656, the target gene of miR158 in Chinese cabbage, using RACE technology. Its DNA sequence,2175bp, consists of two exons and one intron, and its ORF sequence,2046bp, encodes a PPR protein with 682 amino acids. BLAST search revealed that the amino acid of Bra027656 was highly similar to that of the PPR-B, which suggested that Bra027656 might have the same function as PPR-B.(3) qRT-PCR analysis showed that pre-Bra-miR158al and pre-Bra-miR158a2 had different expression patterns. In general, pre-Bra-miR158al and pre-Bra-miR158a2 had higher expression in inflorescence, but the relative expression of pre-Bra-miR158al was higher than that of pre-Bra-miR158a2, especially in the third stage of flower bud. We also got the same results through promoter-GUS reporting system. Based on these datas, we thought that miR158 might play an important role in pollen development in the binucleate microscope stage and trinucleate microscope stage, and pre-Bra-miR158al plays a major role in the synthesis of miR158 in Chinese cabbage. Sequence alignment showed that there was a big difference between the pre-Bra-miR158al and pre-Bra-miR158a2 promoter sequences, so the temporal and spatial expression patterns differences of them may be caused by the difference in its promoter sequences.(4) We then constructed vectors for over-expression and inhibition of Bra-miR158, and transformed them into Chinese cabbage ’Youqing 49’. Cytological and morphological observation of transgenic plants showed that overe-xpression of Bra-miR158 caused pollen abnormal development. Approximately,25% of the pollens in transgenic plants lost vitality, and this aberration began in uninucleate microspore stage, which caused the pollen contents degradation and pollen shrunken or vacuolization. These results matched with the expression pattern of Bra-miR158, indicating that miR158 has an important role in pollen development process. However, there was no abnormal phenotype in the transgenic plants inhibiting the expression of miR158, so we proposed that the downstream gene of the Bra027656 were not sensitive or had no reaction to the down-regulation of Bra027656. We hypothesized that Bra027656 might work in pollen development process by acting in some important metabolic pathway genes in the mitochondrias of microspore, and these genes’abnormal expression could cause metabolic disorder in microspore inclusions, which would result in the decomposition of inclusions, thereby indirectly affected the synthesis of the pollen wall material and led to pollen shrinkage, collapse or vacuolation.