IC4R005-miRNA-2012-23071571

Project Title

Identification of Novel Oryza sativa miRNAs in Deep Sequencing-Based Small RNA Libraries of Rice Infected with Rice Stripe Virus

The Background of This Project

[[File:|700px|thumb|right|Figure 1. GWA Analysis of Al Tolerance within and across Rice Subpopulations.]]

• MicroRNAs (miRNAs) are small 19–24 nt RNAs that play essential roles in eukaryotes by targeting complementary mRNAs for degradation or translational repression. In plants, primary miRNA (pri-miRNA) is first transcribed by polymerase II, and then processed by Dicer-like 1 (DCL1) into the precursor miRNA (pre-miRNA), normally of about 70–300 nucleotides (nt). The pre-miRNA is further processed into the mature miRNA:-miRNA* duplex [3,4,5]. These processes occur in the nucleus. In the next stage, the duplex is transferred into the cytoplasm and unwound. The miRNA is then assembled into and RNA-induced silencing complex (RISC) and guides the RISC to cleave or suppress the target mRNA. miRNAs in plants regulate leaf morphogenesis, the development of roots and flowers and other key processes, and are recognized as important regulators of plant development. Recent research has revealed that miRNAs also play roles in plant defense against pathogens by regulating the expression of resistance (R) genes directly or indirectly, or targeting the viral genome to impair viral replication. Hence, the miRNA pathway also plays a key role during pathogen-plant interactions. In plants, over 4600 miRNAs have been identified from over 50 species (miRBase version 18.0, http://www.mirbase.org/cgi-bin/browse.pl). Medicago truncatula, Oryza sativa and Glycine max are the three plants that have the most identified miRNAs (respectively 674, 661 and 395 miRNAs). Some miRNA families have functions that are conserved across the plant kingdom and thus their sequences are similarly conserved (e.g. miR156, miR159, miR160 and miR165). Other miRNA families are specific to particular plants, and are not found elsewhere, indicating that they have novel and specific functions.
• icating that they have novel and specific functions. With the development of next generation sequencing technologies, deep-sequencing has provided a powerful high-throughput strategy for identifying novel miRNAs. In this way, hundreds of miRNAs have been identified from Arabidopsis, Brassica rapa, rice, wheat, barley, peanuts, grapevine and other plants. In rice, Sunkar et al identified 23 new miRNAs from three small RNA (sRNA) libraries of control rice seedlings and seedlings exposed to drought or salt stress; six of the new miRNAs are conserved in monocots. Chen et al identified 24 novel microRNA families from rice embryogenic callus, some of which were suggested to function in meristem development [33]. Li et al investigated the H 2 O 2 - regulated miRNAs in rice seedlings and discovered 32 new miRNAs. Peng et al identified 43 novel miRNAs from the sRNA libraries of rice spikelets [35], while Wang et al identified 75 novel miRNAs from the developing pollen of rice.

Plant Culture & Treatment

• RSV-infected rice were prepared as described [39]. Briefly, viruliferous adult brown planthoppers (Laodelphax striatellus Fallen) (carrying the RSV-Zhejiang isolate) were transferred onto healthy rice seedlings (Oryza sativa L. japonica. cv. Nipponbare) at the three-leaf stage for virus inoculation. Control seedlings were inoculated with non-viruliferous planthoppers. After 72 h, the planthoppers were removed. Systemic infections were confirmed by RT-PCR specific for RSV Zhejiang isolate. One week after inoculation, leaves were collected from the infected and control (Mock) plants, frozen and stored at 280uC until used. Allriceplants were grown in a glasshouse at 28–30uC day/25uC night, with a 12 h day/night light cycle under well-watered conditions.

Research Findings

[[File:|700px|thumb|right|Figure 2. Haplotype analysis of the Nrat1 gene region.]]

Labs working on this Project

• State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Ministry of China Key Laboratory of Biotechnology in Plant Protection, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
• Plant Protection College, Yunnan Agricultural University, Kunming, China
• College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

Corresponding Author

• Jianping Chen(jpchen2001@yahoo.com.cn)