Targeting MIR408 Genes In Wheat With CRISPR/Cas9 Gene Editing Technology

Wheat（Triticum aestivum L.）is one of the important food crops over the world.At present,domestically grown wheat in China can be divided into two types:spring wheat and winter wheat.Since most of the winter wheat varieties cannot survive under the winter climate conditions in the Northeast of China,their promotion in the Heilongjiang Province is restricted.Dongnongdongmai1（Dn1）is the first winter wheat variety that can survive the winter in high-cold areas and the rate of restoring growth in spring is greater than 85%,so it is a precious material for research on crop cold resistance.MicroRNAs（miRNAs）is a kind of endogenous small non-codingRNA with a length of 20-24 nucleotides,which plays an important role in the response of plants to abiotic stress.miRNA is encoded by the MIR gene,and can regulate the signal network of plants at the molecular level by interacting with its target genes,so as to respond to external stress to reduce damage.Therefore,miRNA has received more and more attention as the target for crop genetic improvement.miR408 is a highly conserved miRNA widely distributed in plants.Preliminary laboratory studies have shown that miR408 was involved in the response and regulation of winter wheat Dn1 to low temperature stress.Gene mutation is one of the important methods for studying plant gene function and crop gene improvement,and gene editing technology can generate mutations at specific sites in the plant genome to speed up the process of related research.Compared with gene editing technologies such as ZFN and TALEN,the CRISPR/Cas9 technology that has been developed in recent years has the advantages of high efficiency and relatively simple operation.Therefore,CRISPR/Cas9 technology has been widely used in the study of plants.This study used Agrobacterium to transform callus induced from wheat mature embryo,and used CRISPR/Cas9technology to target the MIR408 gene of winter wheat Dn1,in order to explore the possibility of using CRISPR/Cas9 technology to target wheat non-coding genes.The method laid the foundation for the functional research of wheat miRNA through the method of gene knock-out.The results of this study are as follows:（1）According to the sequence information of TaMIR408-A,B,D and miR408 provided in GenBank,the genome location and conservation of TaMIR408 were determined.Pre-miR408secondary structure prediction results showed that the structure of miRNA:miRNA*region was not obvious.Therefore,the mature miRNA conserved region targeting TaMIR408 was selected,and three sgRNAs targeting TaMIR408-A,B,and D genes were designed:miR408-sgRNA1,2,3,bioinformatics analysis results showed that the GC content of the three sgRNAs was 65%,and all the sgRNAs had a stem loop RAR,2,and 3 secondary structure.（2）Two CRISPR/Cas9 vectors（pTKC1.2 and pBUE411-TaU3p）with single sgRNA were constructed.The construction method of pTKC1.2 was that firstly synthesizing the sgRNA expression cassettes initiated by the Ta U6 promoter with the method of overlap PCR,and then connecting the sgRNA1,2,3 expression cassettes to the pTKC1.2 vector after digestion with Pme I by the one step clone method;pBUE411-TaU3p construction method was that firstly annealing the sgRNA1 single-stranded DNA primer to synthesize the sgRNA guide sequence with sticky ends,and then ligating the pBUE411-TaU3p vector after digestion with Bsa I by T4 ligase.（3）We designed specific PCR primers for cloning DNA fragments containing TaMIR408-A,B,and D respectively,and selected appropriate cloning primers through PCR detection,and ligated the target fragments to the cloning vector for sequencing.The results showed that the sequence of MIR408 in winter wheat Dn1 is the same as the sequence in the China Spring Database.（4）We connected the T7 promoter with the DNA sequence encoding sgRNA and transcribed in vitro to obtain sgRNA;the in vitro enzyme digestion reaction of sp Cas9/sgRNA showed that miR408-sgRNA1,2,3 can target the TaMIR408 genes in vitro,and the activity of each sgRNA was similar.（5）We used mature embryos of winter wheat Dn1 for callus induction,and used Agrobacterium containing pTKC1.2-sgRNA1,2,3 to transform fresh callus for 3-4 weeks,and then we made the callus co-cultivate for 5 days,and resume culture for 14 days.Then we induced the regeneration of callus.By detecting the Cas9 expression cassette,a total of 3 miR408-sgRNA1 T₀ transgenic plants were identified,indicating that the Agrobacterium-mediated transformation method can introduce the CRISPR/Cas9 system into wheat.Although the transformation process of wheat usually produced chimeric plants,the result of gene editing has not been detected in the T₀ generation.At present,the regenerated plants have been transplanted into the field in order to obtain the T₁generation plants for further detecting.