Cloning Of Mirnas From Chlamydomonas Reinhardtii Under Iron Deficiency And HO-1Vector Construction And Transformation

MicroRNAs (miRNAs) are a newly discovered class of endogenous non-protein-coding small RNAs with21nt long that have the ability to base pair with their target mRNAs to repress their translation or induce their degradation in both plants and animals. Growing evidence has demonstrated that miRNAs play multiple roles in a variety of biological processes, including development, cell proliferation and stress responses. Recent studies showed that several miRNAs regulate plant adaptation to sulfate, phosphate, nitrogen and copper deficiency, respectively. However, whether miRNAs are involved in regulation of response to iron deficiency is unknown. Thus far, the major approach for identification of miRNAs is direct cloning and bioinformatics. The green alga Chlamydomonas reinhardtii, a unicellular eukaryote has many advantages, such as a short life cycle, strong adaptability, easy to culture, clear genetic background. Chlamydomonas is similar to higher plants in many aspects including cell structure, metabolism and genetics. Therefore, the single-cell Chlamydomonas is often used as an ideal cell for investigation. It has been reported that Chlamydomonas also contains multiple miRNAs. In this study, we constructed a library of small RNAs from Chlamydomonas reinhardtii under iron-deficieny. Sequencing457clones of the library and subsequent analysis revealed86small RNA and1previously reported cre-miRNAs. Additionally, we analyzed cis-regulatory elements upstream of49of all known miRNA genes in Chlamydomonas and found12miRNA genes containing FeRE1/FeRE2-Like motifs in their promoter regions. Transcriptional analysis using RT-PCR showed that6of the FeRE-containing miRNA genes were up-expressed in response to iron-deficiency.Heme oxygenase-1(HO-1) catalyzes heme catabolism. Recently, HO-1is shown to play multiple roles in biological processes, but the accurate functions of its gene are largely unknown. In this study, we constructed an expression vector harboring HO-1from Chlamydomonas reinhardtii. Digestion with Spe Ⅰ and Bgl Ⅱ, DNA sequencing, GUS staining and PCR detecting proved that the HO-1gene has been cloned into the expression vector. Chlamydomonas nuclear transformation commonly adopts glass beads or particle bombardment. However, these methods are not easy to operate and the transformation rate is low. We transformed Agrobacterium-mediated pCAMBIA1304-HO-1into Chlamydomonas cells. RT-PCR results indicated that the reorganized vector has been successfully introduced to the cell. The result of the present study will provide basis for the further research on functions.