2.1. In silico search for the miR156 homologue and its target genes in the B. vulgaris
The sequences of previously known miR156 precursor gene of eight different plants (Ananas comosus, Arabidopsis thaliana, Camellia sinensis, Ceratopteris thalictroides, Citrus sinensis, Musa AAB, Oryza sativa and Zea mays) were compared at the National Center for Biotechnology Information (NCBI) GeneBank and miRBase database. When we started the first part of our research, the sugar beet KWS2320 genotype was the only genome which was annotated in NCBI, so, refseq-genomic sequences of this plant associated with the miR156 precursor sequences from each mentioned plant were identified through the BLASTN. If there were several similar refseq-genomic sequences with the miRNAs precursor sequences, each of them was individually considered. De novo assemblies of the sugar beet refseq-genomic sequences and precursor miRNA sequences from mentioned plants were carried out using the SeqMan Pro NGen (DNAStar, Inc., Madison, WI, USA). After the alignment, common sequences were considered as the miR156 and miR172 precursors in the sugar beet. Verification of the identified sequences was performed using the BLASTN in other plants' genomes. To identify the complete sequence of the genes in the sugar beet genome, various parts of the refseq-genomic sequences containing the gene sequences were thoroughly investigated via alignment. At last, the miRNA sequences were identified in precursor sequences, and were aligned using the MegAlign Pro NGen (DNAStar, Inc., Madison, WI, USA).
For evaluation of main targets of the miR156 in sugar beet, the sequence of identified SPLs (SPL4 and SPL9) genes was first extracted from the NCBI GeneBank. To ensure that these genes are the target sequences, and to identify the restrictions and complementary areas, two psRNATarget (https://plantgrn.noble.org/ psRNATarget) and the RNAhybrid (http://bibiserv2.cebitec.uni-bielefeld.de/rnahybrid) software programs were used.
2.2. Plant materials and growth conditions
The seeds of Beta vulgaris ssp. Maritima (collected from the Shoush area of Iran), was obtained from the Sugar Beet Seed Institute (Located at Karaj, Iran), and was used in genetic transformation for all experiments. The seeds and plants were cultured in the Murashige and Skoog medium, under the long-day conditions with 16 h light/day and 24/16°C day/night rhythm in the growth chamber.
2.3. The gene construct and transformation
A 127-bp stem-loop fragment of BvmiR156 precursor gene was amplified by the polymerase chain reaction (PCR) with the MIRF and MIRR primers (all primer sequences are given in Supplemental Table 1) introducing the NcoI and BamHI restriction sites which were designed based on the miR156 precursor identified in Beta vulgaris genome. After the sequencing, the P35S:BvmiR156 was generated by replacing the Gus gene with the BvmiR156 gene between the NcoI and BamHI restriction sites in the pGSA1285 vector. The pGSA1285-miR156 construct was sequenced and transformed to the Agrobacterium tumefaciens GV107 strain competent cells using the freezing ⁄heating shock method (Sambrook and Russell 2001). After preparing the beet leaf explants (Mirzaei-Asl et al., 2010), they were infected for five min and incubated at 25 °C for 48 hours on coculture medium (MS medium and 1 mg/l 6-benzyl aminopurine), followed by incubation in selective medium (MS medium, 1 mg/l 6-benzyl aminopurine, 100-150 mg/l Kanamycin, 200 mg/l Cefotaxime). Thereafter, Kanamycin-resistant shoots were appeared in the selective medium.
2.4. The analysis of transgenic plants
The genomic DNA was extracted from the leaves of both regenerated and non-transgenic plants (control) by the CTAB method (Murray and Thompson, 1980). To confirm the transgenicity of the plants, the PGSAF and PGSAR primers (Supplemental Table 1) designed from the pGSA1285 vector promoter and terminator, were used in PCR. The reaction conditions were included the pre-heating of 94 °C for five min, 35 cycles at 94 °C for 30 s, 58 °C for 45 s, 72 °C for 45 s, and the final extension of 72 °C for seven min, for the PCR product sequencing.
2.5. Expression analysis of the miR156 and target genes
The total RNA was isolated from the young leaves of transgenic and non- transgenic plants using the RNX-Plus solution (Sinaclone Co.) according to instruction. Then, it was treated with the RNase-free DNase enzyme (Fermentase Co.) as described in the instruction. Amount of 500 ng RNA was used for the reverse transcription of the miR156, SPL4, and SPL9 by cDNA Synthesis Kit (Fermentase Co.). The RT-PCR reaction was performed with general primers of UB-quinitine (UbiF and UbiR) and actin-8 (ActinF and ActinR). The reaction conditions were included pre-heating of 94 °C for five min, 35 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 45 s, and the final extension of 72 °C for five min.
The quantitative RT‐PCR of the miR156 gene and the target genes (SPL9 and SPL4) were done using the SYBR‐Green PCR Mastermix (Amplicon) and an applied biosystem Roche LightCycler 480 instrument. The Actin 8 gene was used as the internal control. The qPCR reactions were performed with three biological and technical replicates, separately. The PCR conditions were 95 °C for five min followed by 35 cycles at 94 °C for 30 s and 58 °C for 30 s. Transcript levels of the miR156 and the target genes were analyzed using relative quantification by the comparative Ct (2−ΔΔCT) method (Livak and Schmittgen, 2001). The primers used for the RT-PCR and qRT-PCR are listed in the supplemental Table 1.
2.6. Flower and root development analysis
After confirming the transgenic plants, their exact flowering time was compared with non-transgenic plants to determine the effect of the miR156 over-expression on flowering time. Also, the root development under the miR156 over-expression effect was evaluated in transgenic plants in comparison with non- transgenic plants.
2.7. Statistical Analysis
Triplicate samples were collected for each transgenic line. The statistical analysis of the miR156, SPL4 and SPL9 genes expression were conducted using one way-ANOVA test after evaluating "the normality residual exam" by the SAS software (SAS Institute, Cary, NC). Also, correlation analysis test was performed using Minitab software. The P values < 0.05 were considered significant.