Identification And Functional Analysis Of Association-mirna In Maize Induced By Banded Leaf And Sheath Blight

The maize banded leaf and sheath blight (BLSB) has become the main disease in the producing area of china in recent years, and critically affected the maize yield and the improvement of quality traits, owing to the spread and the expansion of occurrence of this disease. Therefore, it is an effective way of cultivating broad-spectrum, efficient, stable, safe and disease-resistant corn varieties through identifying and selecting disease-resistant key genes for BLSB and then carries on breeding for disease resistance, combining with traditional breeding strategies and frontier molecular biotechnology to know disease-resistant molecular mechanism and systematically explore the molecular modulation mechanism. miRNAs are a class of small single-stranded non-coding RNAs that range in length from roughly 21 to 24 nucleotides,which play a central role in regulating plants growth and development and responsing to adverse environmental and so on, tlirough directing the cleavage or repressing translation by interfering with expression of plant mRNA So, it is pressing to screen and study the role of the candidate miRNA and its target genes in process of regulation which invovled in disease resistance and then identify its function.In this study, the maize inbred lines R15 (resistance to BLSB) and 478 (susceptible to BLSB) were used as materials, many candidate miRNA were screened under BLSB stress based on the initial research combing with Solexa depth sequencing and bioinformatics method analysis, and expressions of the candidate miRNA and its target genes of BLSB were detected combing with miRNA in situ hybridization, fluorescence quantitative and semi-quantitative and so on. The main results are as follows:1.39 new miRNAs are obtained which belong to 33 miRNA families, of which, there were five new special miRNAs in BLSB treat and 15 new miRNAs in control, moreover, 15 miRNAs were both exhibited differential expression in the above conditions However, 4 new miRNAs of their expression showed no obvious difference.and 147 known miRNAs were obtained which belong to 25 miRNA family,41 miRNAs were expressed different or significantly different, of which,4 miRNAs were differentially expressed,2 miRNAs were differentially up-regulated and 35 miRNAs were differentially down-regulated.2. The new discovered zma-miRNA36, zma-miRNA32 and zma-miRNA28 kept higher conservative in their sequeces compared with the known zma-miRNA156 family, miRNA171a/b family and zma-miRNA 172 family, respectively; So they could be new members of the three known maize miRNA family respectively. 3. Some new miRNA kept different conservation in the different mode plant.4. Target genes of new 39 miRNAs were predicted and classified respectively, which contained 248 target genes which mainly involved in the growth and metabolism, protein transportion, transcription regulation, response to stress, hormone signaling and electronic transfer and so on. Moreover, target genes of the known miRNAs expressed differentially and tended to target to the transcription factors (MYB, TCP, zinc finger protein, GRAS, ARF et al) in BLSB treat condition5. Some miRNAs associated with disease resistion could meanwhile participate in maize growth and development and play important roles in responsing to sheath blight, and, this type of phcnomenons compose overlay network of miRNA regulation associated with disease resistion.6. The 9 important candidated miRNAs were selected and verifed by Q-PCR It is indicated that these miRNAs were almost down-expressed especially significantly expressed in R15 and-treated after 24 hours. Howeever, mill 168a showed apparently up-expressed in the two materials.7. The two important candidated miRNAs is selected and analysised by ISH (in situ hybridization), It is suggested that the infection sites of BLSB in tissue spreaded from outside to inward with the prolong of time and showed their specific expressions in these sites.8. The target genes of the 10 miRNAs were all detected in R15 and 478, and showed great differences in their expression levels under BLSB treated respectively which included AGO1, GRAS transcription factor, ubiquitin hydrolase, TCP transcription factor, serine/thrconine protein kinase, laccase, ARF hormone response factor 1/2, glutathione suifur transferase, MFS drug transport protein, CCAAT transcription factor and so on. In general, the condidated miRNAs up-expressed in different periods after infected BLSB in R15 and 478 and reached to peak in 24 hours while the target genes down-expressed, On the contrary, the condidated miRNAs down-expressed while the target genes up-expressed. Combine with ISH and Q-PCR of miRNAs, the 10 candidate miRNAs negatively regulated their corresponse target genes which associated with disease resistance through their own peculiar disease-resistant ways in R15 and 478, under BLSB treated. It is donstratcd that R15 and 478 possessed different BLSB resistance characteristics becauce the maize inbred lines R15 enriched more target genes of candidated miRNAs than 478.9. These candidated miRNAs invovled in the complicated defence mechanism of maize to resist BLSB stress, which mostly included signal conduct pathway, antioxidatant stress mechanism, self-feedback regulation, transcription regulation pathway, metabolic pathway and related to disease resistance, efflux of toxic substance, and so on.