| Maize banded leaf and sheath blight (BLSB), caused by Rhizoctonia solani Kühn, is one of destructive disease that results in significant yield loss in most maize-growing areas in the southwest of China. To breed new varieties with efficient, broad-spectrum and stable resistance to the maize BLSB, understanding of resistance mechanism is of great importance. As a class of negative regulators in eukaryote organism, microRNA (abbreviated to "miRNA") regulations almost reach to all cellular levels of organism. In this research, miRNAs of maize under the stress of Rhizoctonia solani Kühn by artifical inoculating were cloned from both high tolerant and high infective inbred lines, and the differences of miRNA content between the two lines were assayed, and target genes of miRNA were predicted and analyzed for its function subsequently yet. Accordingly, the role and mechanism of the miRNAs in resistance channels of BLSB were disscussed, which lay the foundation for breeding and improve the resistant varieties in maize by biotechnology breeding. The findings of the research are as follows:1. The small RNA libraries were constructed from high tolerant inbred lines of R15 and high infective inbred lines of YE478. Seventy-nine small RNA(17-27 nt) were selected, of which the majority has the length ranging from 20 to 24 nt. The sequences, with tRNA, rRNA and degraded fragments excluded, were analyzed by mfold program for stem-loop structure. The results showed that 11 small RNAs which the length ranging from 18nt to 23nt could form stem-loop structures. In addition, miR-6 and zea-miR168a were processed from the same precursor, and they were miRNA and its complementary sequences-miRNA*.2. These sequences were first found except zea-miR168a, and 4 of them began with "U" from 5' termination. This is similar to what have been reported in other plant miRNAs. By "blasting" genomes of other plants including rice, wheat and Arabidopsis, conservation of miRNA was identified among them. As a result, 6 miRNA sequences exhibit conservation to a large extent. On the other hand, there is a trend that these miRNAs were more conservative among maize , rice and Arabidopsis than between maize and wheat. 3. Twenty targets were respectively predicted for 10 of the new miRNAs by a series of methods including using of miRU program, searching CSRDB and NCBI databases, and analyzing fuctional domain of amino acid by "pfam". Resultantly, the targets could be grouped into genes related to defense and disease response(20%), genes concerned with primary and second metabolism,(20%), genes related to signal transduction(30%), transcription factor(15.0%) and others involved in protein processing and modification.4. By RNA-tailing and primer-extension RT-PCR, the 11 small RNAs were detected in high tolerance and high infective maize inbred lines, which confirmed their reality in maize. In according to above reasons, the 9 sequences were identified to be new miRNAs in plants, and attributed respectively to 8 new miRNA families.5. Real-time PCR was carried out to detect the expression of the 9 miRNA in both R15 and Ye478 at different time after inoculating the fungi. The result showed that expression of zea-miR168a, miR-2 and miR-6 displayed similar tendency, which were highly expressed in high tolerance lines. On the other hand, the tendency of expression of miR-4, miR-7 and miR-9 were consistent. The content of miRNAs in the sheath of R15 were extremely larger than that in Ye478 except miR-4 and miR-5. However, miR-3 was likely to be highly expressed in stress positions. Yet, the difference of expression of miR-5 between the two lines were not significant.6. By analyzing both distinction of miRNA contents between high tolerant and high infective lines and function of target genes. The relationship between miRNA regulation and the feasible resistance mechanism against maize BLSB is as follows.First, the maize leaf sheath show more sensitive than leaves to the fungi stress. The expression response speeds of miRNAs in R15 was more rapid and strong when compare with Ye478 in the stress positions.Second, as to zea-miR168a, miR-2 and miR-6, the relative expression trend were similar, which implied that they belong to the same class. Similarly, miR-4, miR-7 and miR-9 might belong to the other class. The expression of two types of miRNAs changed under the infection of the fungi, which indicated that they were closely related to the immune regulation of plant disease resistance. Third, miRNA regulation may be the reason of defense response in maize under infecting the fungi. The role of up-regulation of miRNAs in high tolerant lines was improving the ability to prevent the disease from infection or expansion in affected position by regulating the important enzymes and transcription factors in disease resistance process under the diseases infection.Finally, the specific expression of miRNAs under diseases infection might be the results from maize resistance to pathogen stress. In other words, after being infected by pathogen, expression of some protein is disturbed, including enzyme and transcription factors responding for biosynthesis of miRNA, which promotes or inhibits the expression of miRNA in cell, furthermore, the expression of the gene regulated by miRNAs also changed. Ultimately it is reflected in anti-infection or infection of plants.
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