Screening And Identification Of Cadmium-Responsive MicroRNAs And Their Target Genes In Rice(Orvza Sativa)

Rice is one of the most important grain crops.Cadmium(Cd)is a toxic heavy metal,which can be absorbed and accumulated by rice,affecting the development of growth in rice and even doing harm to human health via food chain.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs,which play crucial roles in plant growth,development,stress response,hormone response and many other biological processes.The research of miRNA in rice Cd stress response will help reveal the regulation mechanism of rice response to Cd stress at the posttranscription level,provide valuable information for breeding low Cd rice varieties,and discover stress resistance genes and genetic improvement.But at present,reports on Cd-responsive miRNAs and their target genes are limited,updated high-throughput sequencing and degradome sequencing techniques make it possible to identify novel Cd-responsive miRNAs and their target genes more accurately.Nipponbare(Oryza sativa cv.Nipponbare)were selected as the research materials.Threeweek-old seedlings were treated with Cd(50 μM),and Cd-free treatment was used as a control.After 3 days of Cd treatment,the roots of these two groups were collected to extract total RNAs for miRNA high-throughput sequencing and degradome sequencing.Then the miR408-3p and its target gene UCL7,which were significantly differentially expressed under Cd stress,were selected for transgenic research on rice over-expression and CRISPR-Cas9 knockout.The results of this research were as follows:1.Six small RNA(small RNA,s RNA)libraries were constructed from the Cd-treated and control groups,with three biological replicates in each group.With the help of miRNA highthroughput sequencing,we totally identified 2,369 miRNAs including 1,589 novel miRNAs,of which 132 significantly differentially expressed miRNAs in Cd-treated and the control rice roots were identified.Moreover,17 miRNAs with the most significant difference in expression between Cd-treated and the control were selected and their expression levels were investigated using quantitative real-time PCR(q RT-PCR),15 out of these 17 miRNAs had the similar results compared with the results from high-throughput sequencing.2.Two degradome libraries were constructed from Cd-treated and control for sequencing,534 differentially expressed target genes and cleavage site from 49 miRNAs were identified out of these 132 significantly differentially expressed miRNAs.Target gene functinal analysis showed that these target genes were mainly involved in metabolic process,protein binding,regulation of transcription and stress response.Meanwhile,4 miRNAs and their target genes were selected for q PCR verification,the results showed that the expression of the 4 groups of miRNAs and their target genes were consistent with the sequencing,and the expression of miRNAs and target genes were negatively regulated.3.MiR408-3p and its target gene UCL7,which were significantly differentially expressed under Cd stress,were selected for transgenic research with rice over-expression and CRISPR-Cas9 knockout.Thirty miR408-3p and UCL7 overexpressing plants were obtained,then 11 transgenic plants were selected for hygromycin gene and UCL7 fluorescent protein identification.10 miR408-3p and 9 UCL7 overexpressing plants were identified.At the same time,10 miR408-3p and 8 UCL7 knockout plants were obtained.q PCR analysis and sequencing verification showed that all of them had targeted editing results at the target site.This study provides valuable information for the functional characterization of miRNAs and their targets in regulatory network responsive to Cd stress in rice,and it also offers the important genetic material for the functional study of miR408-3p and its target gene UCL7.