Functional Identification Of AtmiR396 Based On An Efficient CRISPR/Cas9 Technique

miR396 is a class of endogenous non-coding single-stranded small RNA in plants and highly conserved,which exerts regulatory effect on the expression of its target genes at the transcriptional or post-transcriptional level,and participates in many physiological and biochemical responses,such as plant growth and development,morphogenesis,hormone response and biotic and abiotic stresses and so on.In order to explore the effect of AtmiR396 on the growth and development of Arabidopsis,wild type Arabidopsis?Col-0?and AtmiR396 homozygous mutants which obtained using CRISPR/Cas9 gene site-editing technology were used as the research objects in this article.The function of AtmiR396 in Arabidopsis was preliminarily studied by observing the phenotype,measuring the activity of antioxidant enzymes,comparing the expression differences of its target gene GRFs and related gene CKXs,and analyzing the DEGs.The main content and results were as follows:1.Two sgRNAs were designed according to AtmiR396a and AtmiR396b,and were amplified by PCR,the amplified fragments were 500-750 bp,consistent with the expected length of 592 bp.The pHDE-mCherry plasmid linearized by the digestion with Bsa1 was used to assemble the desired band,became recombinant plasmid pHDE-mCherry-AtmiR396,and it was transformed into the DH5?competent cells,and the positive transformed cells harboring the pHDE-mCherry-AtmiR396 construct were confirmed by PCR amplification of the 592 bp fragment.The plasmid in transformed cells was isolated and identified also by PCR.The transformed clones were further sequened to confirm the successful construction of pHDE-mCherry-AtmiR396 expression vector.2.The recombinant plasmid pHDE-mCherry-AtmiR396 was then introduced to Agrobacterium GV3103 competent cells.The positive transformed Agrobacterium was identified by PCR and sequencing and used to transform Arabidopsis thaliana.3.The T₀ seeds that carried fluorescence were screened to obtain T₁ plants,which were identified and confirmed for AtmiR396 gene knockout and Cas9 integration,AtmiR396 Homozygous mutants were obtained.The T₁ seeds without fluorescence were selected to obtain T₂ plants by PCR and sequencing check,the external conversion element was separated successfully and obtained AtmiR396homozygous mutants which can stable inheritance.4.By tracking the AtmiR396a mutant and wild type seedlings for 10 consecutive days,it was found that the root length of AtmiR396a mutant was significantly longer than that of wild type,and the growth and development phase of the mutant was shorer than that of wild type.Microscopic observation revealed that the root tip root-hair zone of wild type plants had almost no root hair,however the root hair zone of the AtmiR396a mutant was long and dense.However,there was no significant difference between AtmiR396b homozygous mutant and wild type throughout the growth cycle.5.Compared with the wild type,the SOD activity,POD activity,CAT activity,and PPO activity in AtmiR396a mutant seedlings increased,indicating that AtmiR396a mutant seedlings can remove reactive oxygen species produced by plants due to external environmental influences,have good adaptability to the environment and shows better vigor and growth potential.6.Differential expression analysis of target gene GRFs mediated by AtmiR396a showed that the expression of GRF1,GRF3,GRF7,and GRF9 increased,with the decrease of GRF4 and GRF8 and no change of GRF2 after the knockout of AtmiR396a gene.Among them,GRF3 was most significantly increased relatively.Compared with previous studies,it can be determined that AtmiR396 might mainly affect the growth of Arabidopsis roots by upregulating GRF3.The results also showed that the expression of CKX3 and CKX4 decreased,and the expression of CKX7 was not significantly different after AtmiR396a gene was edited.It proved that AtmiR396a can influence the expression of CKXs.7.Through RNA-seq,a total of 1461 DEGs in the roots of AtmiR396a mutants were obtained,including 1082 up-regulated genes and 379 down-regulated genes.The heat map analysis of the DEGs in the roots of AtmiR396a mutant and wild type Arabidopsis showed that there was a significant difference in clustering between them both.The GO and KEGG enrichment analysis of DEGs in the roots of AtmiR396a mutants showed that AtmiR396a mainly transmits signals through transmembrane transport and affects the growth of plant roots through signal transduction and hormone response.During the growth and development period of Arabidopsis seedlings,the differential expression genes in AtmiR396a mutant roots were mainly related to gibberellin.