Growth of chlorophyll cells of B. gracilis

The cell line was developed in INIFAP-Celaya¹³. An inoculum of chlorophyll cells Bouteloua gracilis (HBK) Lag added. ex Steud., TIANSJ98, 25 ml of liquid medium MPC, the basal medium containing Murashige-Skoog plant, 2,4-D, BAP, adenine and sucrose at pH 5.8. Cultures were maintained under stirring at 90 rpm, with continuous fluorescent light (77 μmol^-1 m^-2) at a temperature of 33 ± 1 °C for 5 d. Subsequently, cells were harvested by filtration and the excess medium was removed. They were frozen in liquid nitrogen and stored at -70 °C until used.

Extraction of total RNA

Approximately 150 mg of B. gracilis cells were lysed by maceration with the Sample Grinding Kit™ (GE Healthcare, Sweden) system and subsequently RNA was isolated using the extraction method Trizol™ (Ambion, USA) and Plant RNA Isolation Kit™ (Ambion, USA) according to the manufacturers specifications.

Cloning of sRNAs

The cloning of the sRNAs by concatemerization was performed with the miRCat™ (Integrated DNA Technologies, USA) kit according to the manufacturer’s recommendations. Concatemers obtained were cloned into the PCR 4-TOPO (Invitrogen™, USA) vector for later insertion into chemically competent bacteria E.coli TOP10 One Shot™ (Invitrogen, USA). From the transformed cells, by extraction of plasmid DNA by the alkaline lysis miniprep was performed. SRNAs sequences inserted at the site of the polylinker of four PCR-TOPO vector was amplified with T7™ (5’-TAA-TAC-GAC-TCA-CTA-TAG-GG-3’) and T3™ (5’ oligonucleotides -ATT-AAC-CCT-CAC-TAA-GA-AGG-3’) flanking the cloning sites of the vector. The amplification program used a 94 °C initial denaturation for 1 min, 35 cycles of denaturation at 94 °C for 1 min, alignment at 55 °C for 1 min, extension at 72 °C for 1 min and at the end of the cycles, a final 72 °C extension for 10 min. The PCR products were separated on agarose gel 1.5% and the bands 190 to 400 base pair (bp) were recovered from the gel and purified using minicolumns by Wizard SV Gel and Clean- Up System according to fabricant specifications. The purified DNA was amplified and sent for synthesis and DNA sequencing at the Institute of Biotechnology of the UNAM located at Cuernavaca, Morelos, Mexico. Sequencing was performed on a Perkin Elmer Model 3730 (Applied Biosystems, USA).

Bioinformatics analysis

The obtained sequences were downloaded for FASTA alignment analysis and by using the BLASTn program through NCBI platform (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the algorithm for alignment two or more sequences, and preset parameters for an alignment of high similarity (MegaBLAST). Sequences corresponding to vector were eliminated, as well as the connector (5'-ACC-TTG-CCT-GTG-ACA G-3') that are present in sequences possible miRNAs.

The candidate sequences of 18 to 25 nucleotids were analyzed in the miRBase¹⁴ database and compared with mature miRNAs Viridiplantae kingdom there reported, according to the algorithm and preset parameters. Candidate sequences were also analyzed in the UEA Workbench¹⁵ with platform miRCat tool for the precursor sequence and secondary structure. They were used as reference genomes of Oryza sativa, Zea mays, Sorghum bicolor and Glycine max with preset parameters. Moreover, all sequences were compared to the Rfam¹⁶ database for possible annotations within the different types of RNAs (rRNAs, tRNAs, mRNAs, ncRNAs, etc.) with preset parameters.

Finally, the sequences similar to known miRNAs in order to find target mRNA was carried out using the psRNATarget¹⁷ program. Because the genome of Bouteloua gracilis has not been reported, the database annotation of transcripts rice MSU version 7¹⁸ was used. The parameters used for searching for target mRNAs were more restrictive than the default values, “E value” (maximum expectation) maximum used was 2.0 (3.0 default) and “UPE” value (accessibility to the binding region) up to 20 (25 default); by decreasing the maximum values of both parameters fewer false positives are obtained¹⁷. Sequences of potential target genes were downloaded, and functional analysis of these, with Blast2GO program¹⁹ was performed.

Identification of miRNAs in Bouteloua gracilis

Ten (10) plasmids were amplified, four were selected to generate PCR products ranging from 400 to 150 nucleotides. Sequencing and analysis of the amplified DNA showed the existence of nine sequences with lengths of 18 to 25 nucleotides of the miRNAs characteristic (Table 1). These sequences were analyzed on the basis of data Rfam observed lack of similarity with reported there RNAs (rRNA, tRNA snRNA, snoRNA). Analysis of sequence homology with the miRNAs deposited in miRBase¹⁴ showed that sequences 1 to 4 had the best alignment with the miRNAs miR7828-5p of Populus trichocarpa²⁰; the miR319 reported in Brachypodium distachyon²¹, Oryza sativa²², Sorghum bicolor²³ and Zea mays²⁴; the miR397-3p from Malus²⁵ and the miR397- 5p reported in Linum usitatissimum²⁶ and Brachypodium distachyon²³. However, five to nine sequences did not show similarity to previously reported miRNAs.

In order to characterize these sequences, the UEA miRCat Workbench tool¹⁵ was used which is a platform for predicting new miRNAs, as well as for performing mapping studies of potential miRNAs in a genome of interest. If the mapping of such genome is positive, it allows for determining whether the miRNA precursor sequence has a stem -loop (pre -miRNAs) secondary structure. The analysis was performed with all the obtained sequences and the results demonstrated that only miRNAs and characterized (miR397 and miR319) generated pre-miRNAs. So the sequence two (miR319) was located in the genomes of Zea mays, Oryza sativa and Sorghum bicolor where the structure of the pre- miRNA (Table 2, Figure 1) was obtained. With respect to sequence four (miR397-5p) it was also possible to identify their secondary structure, but only in the genome of Glycine max (Table 2; Figure 2). Regarding sequences one, five, six, seven, eight and nine, none could be identified in any of the reference genomes.

Figure 1 Secondary structure bioinformatically predicted of pre-miRNAs for sequence two (miR319-3p). Green indicates the miRNA sequence identified. A) Location of chromosome one of Oryza sativa. B) Location of chromosome three of Sorghum bicolor. C). Location of chromosome three of Zea mays 

Figure 2 Secondary structure bioinformatically predicted of pre-miRNAs for sequence for four (miR397-5p). In green the miRNA sequence identified is shown. A) Location of chromosome eight of Glycine max. B) Location of chromosome 13 of Glycine max 

MRNA target identification

Using the psRNATarget¹⁷ program, predicted target mRNAs were used to identify potential targets of all sequences based on the degree of complementarity and the ability to access the complementary region. The analysis showed that only miR397-5p predicted six possible target mRNAs, which encode proteins laccases (Table 3). Functional analysis of target genes showed that encode proteins involved in metabolic processes and catabolic processes of lignin phenylpropanoids. In addition to these functions, each gene has different annotation involved in other processes. Five (Os01g63190, Os01g63200, Os05g38420, Os11g48060 and Os12g15680) have copper binding annotation. Two (Os01g63200 and Os12g15680) participate in the development of the reproductive system. While Os11g48060 has functions involved in cell wall formation and Os05g38420 is involved in responses due to lack of water.