Research Of MicroRNA Between Gossypium Harknessii Cytoplasmic Male Sterile Line And Its Maintainer Line

Cytoplasmic male sterility (CMS) cotton has significant advantages in utilizing heterosis and hybridization. Currently, the molecular mechanism of CMS in cotton is still not clear because of the limitation of cotton genome information. miRNAs represent an extensive class of endogenous non-coding small RNAs. They play important roles as negative regulation factors in gene post-transcriptional expression of eukaryotic organisms. miRNAs play vital roles in the gene regulation at various stages of plant development, such as organogenesis and morphogenesis, hormone secretion, signal transduction and stress response. In this research, in order to screen the new cotton miRNAs and the differentially expressed miRNAs and their target genes between CMS cotton and its isogenic maintainer, the buds of GossyPium hirsutum CMS cotton and its isogenic maintainer at microsporocyte stage were harvested and then sequenced by the technology of Solexa.The main results and conclutions are as following:1. By Solexa sequencing, in the CMS cotton ZBA, totally 9 823 976 small RNAs were obtained, including 3 712 462 miRNA sequences, after removing redundant sequences, 145 unique miRNAs were obtained, it contains 92 miRNAs belong to 37 miRNA families and 53 candidate cotton miRNAs. In the maintainer ZB, totally 11 449 151 small RNAs were obtained, including 4 450 631 miRNA sequences, after removing redundant sequences, 151 unique miRNAs were obtained, it contains 90 miRNAs belong to 38 miRNA families and 61 candidate cotton miRNAs. In all, 10 new cotton miRNAs were found in the two materials.2. The results of target gene predication and bioinformatics analysis implied that the miRNAs we identified here were mainly regulate the process of energy metabolism, metabolism, biotic or abiotic environmental stress response, hormone secretion, signal transduction,organ and tissue development, transcriptional regulation. And the processes were realized mostly by regulating the transcription factor.3. By statistical analysis, 42 differentially expressed miRNAs belong to 21 miRNA families were isolated between the two materials.4. Among the predicted target genes of differentially expressed miRNAs, some transcription factors related to flower development and male gametogenesis were found. Such as AUXIN RESPONSE FACTOR (ARF), F-box, Ubiquitin, WRKY, APETALA2 (AP2)-like, SQUMOSA PROMOTER BINDING PROTEIN LIKE (SPL), MYELOBLASTOSIS (MYB) and Gibberellin.5. The results of Real-Time RT-PCR furthere confirmed that the miRNA156 which associated with male gametogenesis was differentially expressed between the two materials. Previous researches reported that miRNA156 affects male gametogenesis by retulating the expression of the SPL-like gene.6. With the conserved domain of SPL-like gene, homology search was performed in the cotton EST database. Finaly, 16 potential cotton SPL-like genes were identified. Real Time RT-PCR analysis showed that 5 SPL-like were up-regulated and 5 SPL-like were down-regulated in the CMS cotton ZBA.