Mechanism Of MiR156 Targeting SPLGene Regulating Lateral Root Development In Potato

MicroRNAs(miRNAs)are a class of endogenous non-coding small molecule RNAs that usually regulate the expression of target genes at the post-transcriptional level and play an important role in each stage of plant growth and development.miR156 is one of a class of evolutionarily highly conserved miRNA families,which includes 11 members.The SPL gene is targeted for regulation,which is involved in regulating plant growth and development,hormone response,stress response and photosynthesis.There are few studies on the regulation mechanism of miR156 on potato growth and development and response to drought stress.In this study,the amiRNA and STTM technology were used to construct potato miR156 overexpression vectorand and silence expression vector.The potato cultivar "Desiree" was transformed by an Agrobacterium-mediated.Based on the qRT-PCR analysis of miR156 and StSPL9 expression in transgenic plants,the biological characteristics of miR156 and its mechanism of regulating lateral root growth and development were studied.It provides a theoretical basis for further studying the miR156/SPLs regulation mode involved in regulating potato lateral root development.The main results obtained in this study are as follows:1.Stu-miR156 bioinformatics analysis showed that there were 11 precursor sequences and processed to form 16 mature sequences.The target gene online prediction tool psRNATarget found that the target gene StSPL9 was targeted and bound by Stu-miR156 at the 1296-1316 bases.2.A plant transient expression vector,pCAM-GFP-SPL9,was constructed,and Agrobacterium-mediated transformation was used to transform B.nigeri.Observe the fluorescence distribution under a laser confocal microscope.The results showed that pCAM-GFP was localized to the cell membrane,nucleus,and cytoplasm,but pCAM-GFP-SPL9 was initially determined to be localized in the cytoplasm and nucleus.3.An amiRNA technology was used to construct a pCPB121-miR156 overexpression vector in potato.A potato cultivar "Desiree" were used as materials.Agrobacterium-mediated transformation was used to transform potatoes to obtain transgenic plants.qRT-PCR was used to analyze the expression of Stu-miR156 and its target gene StSPL9 in the roots,stems and leaves of transgenic plants.The expressionof Stu-miR156 in roots and stems and leaves of transgenic plants was increased.While the expression of the target gene StSPL9 was down-regulation in roots and stems and leaves.Compared with wild type plants,the plant height and root length of transgenic lines were significantly reduced,growth was inhibited,and lateral roots were significantly increased.It shows that miR156 negatively regulates the expression of target genes and participates in potato growth and development.4.Primers were designed according to the stem-loop structure of STTM,BamH? and Hind ? were used as restriction sites.The silencing expression vector Stu-miR156 STTM was constructed and transformed into potatoes to obtain transgenic plants.Design specific primers to detect transgenic plants.qRT-PCR was used to analyze the expression of Stu-miR156 and its target gene StSPL9 in the roots,stems and leaves of transgenic plants.The results show: the expression of Stu-miR156 in transgenic plants was down-regulated;however,the expression of the target gene StSPL9 was up-regulated.This shows that STTM technology can effectively silence the expression of miRNA.5.Each line was randomly selected to count the number of lateral roots and the length of lateral roots.Compared with the wild type control group,the number of lateral roots of the transgenic plants over-expressing miR156 was significantly increased,and the root length and plant height were significantly shorter.However,the number of lateral roots of the transgenic plants that silenced with the expression of Stu-miR156 STTM decreased,the stems became thinner,and the lateral root length and plant height were shorter.It shows that miR156 regulates plant growth and development.