Identification And Functional Characterization Of MicroRNAs Involved In Pollen Development In Brassica Campestris Ssp. Chinensis

microRNAs (miRNAs) are a class of noncoding small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. Pollen development is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. In present, research results about the functions of miRNAs in sexual reproduction, especially in pollen development process, are rare and they are not enough to make clear their regulatory functions and mechanism of action in pollen development process. The’Aijiaohuang’ genic male sterility AB line (ajhGMS ’Bcajh97-01A/B’) in Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis) was used in the present study. Firstly, a microarray assay was conducted on inflorescences of a sterile male line ’Bcajh97-01A’and a fertile male line ’Bcajh97-01B’, in order to screen the differentially expressed miRNAs in the two kind of materials. Secondly, two independent small RNA libraries were constructed from the flower buds of ’Bcajh97-01A’ and ’Bcajh97-01B’. The libraries were subjected to high-throughput sequencing by using the Illumina Solexa system. The aim of high-throughput sequencing was to identify conserved and novel miRNAs in Brassica campestris, meanwhile, a batch of differentially expressed miRNAs would be obtained between the two small RNA libraries. Thirdly, a new technology, degradome sequencing, was used to identify the miRNA targets. At last, more research on the functions of MIR158in pollen development process was done. The main results and conclusions were summarized as follows.(1) A microarray assay was conducted to screen the differentially expressed miRNAs between the sterile male line ’Bcajh97-01A’and the fertile male line ’Bcajh97-01B’.The results showed that44miRNAs were differentially expressed in the two lines. Of these,15had over1.5-fold changes in their transcript levels, with9upregulated and6downregulated miRNAs in inflorescences of ’Bcajh97-01A’.Through computational methods,13members were predicted for3miRNA families (miR158, miR168and miR172) and22genes were predicted to be their candidate target genes. By using5’modified RACE,1target gene of miR158,2target genes of miR168and5target genes of miR172were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the3miRNAs in the flower buds at five developmental stages, and the results were generally consistent with those of the microarray.(2) Two independent small RNA libraries were constructed from the flower buds of ’Bcajh97-01A’and ’Bcajh97-01B’. The libraries were subjected to high-throughput sequencing by using the Illumina Solexa system. Eight novel miRNAs on the other arm of known pre-miRNAs,54new conserved miRNAs, and8 novel miRNA members were identified. Twenty-five pairs of novel miRNA/miRNA*were found. Among all the identified miRNAs,18differentially expressed miRNAs with over two-fold change between flower buds of ’Bcajh97-01A’ and ’Bcajh97-01B’ were identified. qRT-PCR analysis revealed that most of the differentially expressed miRNAs were preferentially expressed in flower buds of ’Bcajh97-01B’.(3) A degradome library was constructed from the inflorescences of ’Bcajh97-01B’.The library was subjected to deep sequencing by using the Illumina Solexa system. Then the identified miRNAs in the high-throuthoutput sequencing and the degraded mRNA sequences in degradome library were used for data analysis. Finally,18target genes of9miRNA families were identified. By using5’modified RACE,3target genes of2miRNA families were demonstrated.(4) MIRI58played important role in pollen development. And its target gene Bra027656, which coded an atypical PPR protein, was likely to function in early stage of pollen development. miR158was an differentially expressed miRNA which was screened by using miRNA array and high-throughput sequencing. We construced35S::MIR158over-expression vector and introduced it into flowering Chinese cabbage (Brassica campestris ssp. chinensis var. parachinensis) by agrobacturium-mediated genetic transformation. Finally, we obtained mir158oe transgenic plants and empty vector control transgenic plants. qRT-PCR analysis showed that miR158was up-regulated and Bra027656was down-regulated in mir158oe plants. The mir158oe and control transgenic plants grew normally and they could bloom and produce pollen. No difference was observed between them. The results of Alexander staining and aniline blue staining of pollen showed that plenty of pollen grains were aborted in mir158oe plants. The results of in vitro pollen germination showed that96.8%of pollen grains from control plants could germinate normally, while only11.4%of pollen grains from mir158oe plants could germinate normally. The results of in vivo pollen germination showed that the pollen germination rate was lower in mir158oe and there were less pollen tube growing through the style, compared with control. Under scanning electron microscopy,90%of pollen grain morphology from mir158oe were abnormal, while99.2%of pollen grain had normal morphology. The above results indicated that the over-expression of MIR158gene in flowering Chinese cabbage led to the abnormal development of pollen. Analysis results of nucleotide sequence and amino acid sequence of Bra027656showed that this gene could code a protein belonging to’P’subfamily of PPR protein family. By using qRT-PCR analysis, we found Bra027656was differentially expressed between flower buds of ’Bcajh97-01A’ and ’Bcajh97-01B’ in early stages of pollen development. Combining the analysis results of MIR158function, we speculated that Bra027656was likely to play role in early stages of pollen development.