Identification Of Stress-regulated MicroRNA In Cotton

MicroRNAs (miRNAs) are a class of small, endogenously non-coding RNAs that are recently found to be negative regulators of gene expression in eukaryotic organisms. Plant miRNAs play key roles in a wide range of biological processes, including meristem cell identity, organ polarity, and developmental timing as well as in response or tolerance to biotic and abiotic stress. Thus, the research on stress-regulated miRNAs could help us gain a better understanding the mechanism and the pathway of gene regulation in plant tolerance to stress, and provide new information for transgenic breeding.Cotton (Gossypium hirsutum L) is the foremost fiber crop in the world. However, litter is known for the stress-regulated miRNAs in cotton. In this study, the cotton plants treated with high salt stress and infected Verticillium dahliae were used as materials. Using bioinfomatic approach and experimental technology, the stress-regulated miRNAs and their targets were identified in cotton. The profiling of these miRNAs lay the foundation for further investigating their functional mechanism in the regulation of stress-defence responses, and the main results were as follows:1. In plants, 42 conserved miRNA families were sorted out from the miRBase database (Release 10.1). Their sequences were used as references in homology-research method for miRNA prediction. In addition, EST, GSS and Core-Nucleotide sequences were used as the miRNA sources to advance the method for miRNA prediction.2. Using the advanced method, we predicted new miRNAs in cotton, tomato and wheat. Of the predicted miRNAs in cotton, two were from GSS sequences; three were from Core-Nucleotide sequences; the remaining sixteen were from EST sequences.3. MiRNA microarray was used to confirm the predicted miRNAs in cotton. The results showed that 10 miRNA families had signal. Using RT-PCR, the profiling of these miRNAs was investigated in Shannong91-11 (salt-tolerant cultivar) and Lumian6 (salt-sensitive cultivar). The result showed that miR156a, miR156d, miR156e and miR169 were down-regulated by salt stress in Shannong91-11, while miR167a and miR399a were up-regulated; only miR159 were down-regulated by salt stress in Lumian-6.4. Using bioinfomatic approach, 23 target gens were predicted. Their profiling was further investigated and showed that SPL3,RNA helicase and HAP2 were up-regulated by salt stress in Shannong91-11, while ARF8 and Ghi.6739 were down-regulated. 5. Four small RNA libraries were constructed from mocked and infected roots of two cotton cultured species which are with different Verticillium wilt tolerance ('Hai-7124', G. barbadense L., a Verticillium-tolerant cultivar, and'Yimian-11', G.ossypium hirsutum L. a Verticillium-sensitive cultivar). A total of 68, 970, 173 alignable small RNA reads were obtained by high-throughput sequencing technology. Following a set of strict filtering criteria, a total of 215 miRNAs were detected. The fractions of small RNAs were substantially different between libraries. The sample-specific tags accounted for about 40% of all unique sequence reads in each one of the four libraries.6. The expression abundance of miRNAs in data sets was analyzed by counting the number of transcripts per million (TPM) clean tags in libraries. The results showed that 38 miRNAs were up-regulated in Yimian-11 in response to Verticillium infection, while 15 miRNAs were down-regulated; 24 miRNAs were down-regulated in Hai-7124 in response to Verticillium infection, while 15 miRNAs were up-regulated. In addition, there were 54 and 44 miRNAs with a genotype-specific expression under mocked and infected conditions, respectively. Using bioinfomatic approach, 161 target gens were predicted for the 63 miRNAs. Of them, 25 mRNAs were involved in stress response, resistance and signaling conduction.