IC4R010-miRNA-2010-19796675

Project Title

A class of small RNA molecules called microRNAs (miRNAs) has been identified in recent years. MiRNAs are endogenous non-coding RNAs, are 20–22 nucleotides long, and regulate gene expression in eukaryotes ranging from animals to plants [1–6].
Four Dicer-like enzymes (DCL1–DCL4) are encoded in the genome of Arabidopsis thaliana [20]. It has been shown that DCL1 is involved in miRNA accumulation [21]. However, there is no dcl1 mutant available for rice. A recent study has shown that the loss of function of OsDCL1 transformants could be used to identify miRNAs[22].
MiR399 was shown to down-regulate UBC24 mRNA accumulation and to be involved in plant responses to Pi starvation in planta [24,25]. MiR393 has also been shown to inhibit the expression of TIR1 to down-regulated auxin signaling and seedling growth under abiotic stress conditions [4,26]. Moreover, miR159 was shown to involved in hormone signaling and dehydration responses in Arabidopsis [27,28]. However, these observations were all shown in Arabidopsis, few stress-related miRNAs have been discovered in rice.Among miRNAs discovered in rice, only two have been found to be related to abiotic stress, miR393 and miR169g, both up-regulated by dehydration [29].

The seeds (O. sativa L. ssp. Japonica cv 9522) were stimulated to break dormancy and germinated. Uniformly germinated seeds were sown in plates, immersed in water for 1 d at room temperature and 1d at 37 °C, and then transferred to grown chamber with a 16-h light and 8-h dark photoperiod at 25 °C. Two-week-old seedlings were treated with 100 μM ABA, 4 °C, dehydration (exposed to dry air), or 300 mM NaCl individually for 24 h. Untreated seedlings were used as controls. Entire seedlings were collected and immediately transferred into liquid nitrogen. The frozen seedlings were used for generation of small RNA libraries, the miRNA array and for Northern hybridization.

To identify novel miRNAs from rice, we generated five independent small RNA libraries with size range of 18–28 nucleotides from untreated 2-week rice seedlings and 2-week rice seedlings treated with ABA, 4 °C, dehydration, and NaCl, respectively. After sequencing, about 3900 individual small cDNA sequences between 18 and 28 nucleotides in length were isolated (Table 1).

National Centre for Plant Gene Research, Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,P.O. Box 2707, South 1-3, Zhongguancun, Beijing 100080, P.R. China
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P.R. China
National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China