Function Research Of MiR164 In Rice Growth And Development

MicroRNA are a short chain classes of small non-coding RNA,which can combine the specific site of target gene mi RNA by complementary base pairs,and cleave target mRNA or inhibit the translation process.miR164 is highly conservative in plants and the target genes are NAC transcription factors.In dicotyledonous model plant Arabidopsis?Arabidopsis thaliana?,the regulatory pattern and the molecular mechanism of miR164 has been known,but in rice?Oryza sativa L.?which as a kind of important food crops,miR164s'function has not been clear.In this study,by analysising of the software and website precursor,we got the secondary structure and the target genes of miR164.GUS staining and real-time quantitative PCR were also used to analysis the expression patterns miR164 and its target gene Os12g0610600 in different space-time.Overexpress pre-miR164 by fusioning ubi promoter,construct antisense plants by MiRNA Target Mimics and CRISPR-Cas9technology,and then observed stable and heritable plants'phenotype.Under different expression levels of miR164,by comparing the phenotype of the plant,we found that mi R164 affects on rice growth and development.Transcriptome was also been conducted,to find the possible regulation pathways of miR164,this may provide guidance for the follow-up study of miR164.The results are as follows:1.Bioinformatics analysis showed that miR164 have six loci in rice genome,their mature mi RNA are both 21 nt and relatively conservative sequence.As same as miroRNA precursors,the secondary structures of pri-mi R164 also have the hairpin structure,and the mature miRNA are located on the arm.By constructing transgenic plants which fuse GUS gene with miR164 promoter,and then detecting the expression level of different tissues with real-time fluorescent quantitative PCR,we know that miR164 mainly express in the mature stem,stem nodes and leaves.Moreover,miR164 express in anther and the expression level at the earlier stage was higher than the later stage.By qRT-PCR,the mi R164 is mainly expressed in the leaves and mature anther.2.In order to study the effect of miR164 on the growth and development of rice,mi R164 overexpression plants were constructed,and the expression level of OEmiR164was increased by about twenty times.Compared with the wild type ZH11,OEmiR164plants were shorter,the leaves were curled,and the phenotype may be related to light intensity.The I₂-KI staining rate of over expression plants decreased,the seed setting rate decreased,the length of spikelet became shorter,and the number of secondary branches decreased.The semi thin section observation results of anther development showed that in the S6-S13 stages,the epidermis,endothecium,middle layer and tapetum of some adjacent chambers were not normally separated,most of them only had two to three chambers,and the me trocyte could produce mature pollen grains.3.The transcriptome analysis of OEmiR164 plants showed that there were 498genes with significant difference,297 of them were up-regulated and 201 down regulated.The analysis of phytohormone response pathway showed that the auxin receptor genes,ARF gene and SAUR gene?IAA1,IAA2,IAA17,ARF12,Os10g0510500?had significant changes in the tryptophan metabolism pathway.The indole acetic acid amination synthetase OsGH3 family was up-regulated in both the chip and the young spike.Addention.Additionally,the results of differential genes'transcription factor analysis indicated that the ARF family and NAC transcription factor families were significantly different.4.By using miRNA Target Mimics method we got miR164 down regulated plants,and found that part of the anther chambers became abnormal since S6 stage,and the abnormal chambers can not produce fertile pollen.CRISPR-cas9 plants which had a deletion of the seventh base were also gained,while the phenotype needs ferther study.5.By predicting the target genes of mi R164 from website,we found that Os12g0610600 which belong to NAC transcription factors family have complementary base pairing sequence with mi R164 in its third exon,and its expression level was negatively correlated with the expression level of miR164 in the overexpression and low level expression plants.By constructing transgenic plants which fuse GUS gene with Os12g0610600 promoter,we know that Os12g0610600 mainly express in the mature stem,stem nodes leaves and spikelet.