Preliminary Study On Regulatory Functions Of MiR159 And MiR390 In Rice Growth And Development

Plant microRNA?mi RNA?is a type of small non-coding RNA with a length of about 20-24 nt that has been widely studied in recent years and plays a key role in the regulation of animal and plant gene expression.It is a primary-miRNA?pri-miRNA?transcribed from RNA polymerase II?Pol-II?processed by ribozyme DCL1?DICER-LIKE 1?.Mature miRNAs can be further incorporated into Argonaute?AGO?protein to form an RISC complex?RNA-induced silencing complex?to cleave mRNA of a target gene or inhibit translation of a target gene,thereby achieving negative regulation of expression of the target gene.miRNAs can be paired with the target gene mRNA to regulate target gene expression at the post-transcriptional level by cleaving the target gene mRNA or inhibiting the translation of the target gene,thereby affecting plant growth and development,morphogenesis,metabolism,and stress response.At present,a large number of mi RNAs were found in important food crops and vegetables such as corn,rice,soybeans,cotton,tomatoes,cabbages,cucumbers,etc.These miRNAs greatly affect the yield and quality of crops.For example,the interference of miRNAs in rice on the regulation of target genes,optimization of the rice plant type,making the number of rice tillers decreased,increasing the lodging resistance of rice,and increase the yield of rice;can also be controlled by strigolactone?SL?signaling pathways affect rice tillering and yield;overexpression of miRNAs results in increased number of rice spikes and increased rice grains.This means that mi RNA has great application value and development potential in improving crop traits and developing new varieties with good quality.In this study,we constructed CRISPR/Cas9 and overexpression vectors for six members of miR159 and miR390 and 13 target genes of miR390,and rice was transformed to study the function of miR159 and miR390.The results are as follows:?1?Site-directed mutagenesis of rice miR390 mature sequences using CRISPR/Cas9technology.Mutants were obtained in the T₀ generation which included deletions or insertions of single base A at the editing site,T substitutions to A,and the deletion of three base GCT and six bases AGCTCA,etc.A single base?A-transformed T?homozygous mutant without CRISPR/Cas9 was obtained from the recurrent mutant strain to the T₂ generation,and the rice mutants display phenotypes including increased number of tillers and longer seeds.?2?Temporal and spatial expression of miR390 and its target genes:The results of wild-type RT-PCR showed that miR390 was highly expressed in the above-ground parts of rice,and the expression levels of miR390 and target genes TAS3b1 and06g03970 showed an upregulated trend at the flowering stage,but the expression of ARFs is very low.Whether miR390 regulates the number of tillers and the development of seeds through target genes,and the specific regulation mechanism remains to be further studied.?3?Spatial-temporal expression of miR159:miR159 gene expression is the highest at the mature stage,and miR159f shows an upregulated trend in the expression of miR159f from the bud to the maturation period,both in the aerial and underground parts.During the booting period,the early expression level of miR159a/b/c/d/f was the highest,but miR159e was not expressed at booting stage.From the flowering stage,the expression of miR159c/d decreased from the day of flowering to the sixth day of flowering,and increased at the later stage.By introducing CRISPR/Cas9 technology into rice miRNAs,a series of rice miRNA mutants can be obtained to enrich rice germplasm resources,which can open up new ways for rice genetic improvement and selection of new varieties;CRISPR technology can be used to remove transgenes by hybridization.Plants can be obtained contain plants with no genetically modified components but have been edited and can be stably inherited;these non-transgenic crops do not have risk for genetic safety and will be easily accepted by the community for rapid application.