Study On The Functions Of MiR171a And Its Target Genes By Using CRISPR/Cas9

MicroRNAs(miRNAs),a class of small RNA molecules(?22 nucleotides),impact lots of physiological and biochemical functions of plants.The STTM technology is one of the methods applying in functional analysis of miRNA.Although the miRNA activities could stop by using STTM,it cannot be edited at the genetic level to study the function of individual miRNAs.CRISPR/Cas9 which is target on DNA sequences has been developed as a new technology in recent years,and has been widely used in amounts of plant.In this research,we attempted to create a method and technical system for identifying miRNA functions in Arabidopsis using CRISPR/Cas9 technology,which was also used in rice experiment to determine its fcasibility and its target genes were further studied.The functions of Arabidopsis thaliana miRl71a were identified by STTM and CRISPR/Cas9 separately.Both techniques were found to reduce the expression of miR71a in Arabidopsis thaliana and down-regulate the expression levels of target genes SCL22 and SCL27.The expression level of the target gene SCL6 was up-regulated,the chlorophyll and proline contents were decreased,and the growth potential and drought resistance were weakened.When knocking out miR171a using CRISPR method,a single base insertion mutation was obtained,which will make impact of the activity of miR171a to a certain extent,however the efficiency was not as much as STTM.This result shows that the CRISPR technology is feasible to verify the function of miR171a.In order to explain the function of miR171a in rice experiment,a CRISPR vector containing miR171a dual target targeting miR171a precursor was created,also the rice was transformed by Agrobacterium to gain the knockout strain of miR171a.Among that,the mutant with a deletion of up to 65 bp was gained.When the different mutants were computed,the target gene expression was slightly higher than that of the wild type.The determination of proline,malondialdchydc,peroxidase and soluble sugar before and after the drought treatment was found to be diffcrent from the wild type.However,no significant differences were found during the drought treatment.At the same time,we constructed a CRISPR vector for knocking out HAM1,HAM2 and HAM3 genes respectively,expecting to study the function of individual members of the HAMs family.It was found that when one member was knocked out,the expression levels of the other two would increase.In addition,with the purpose of further validate the function of HAMs not impacted by miR171a,at the early stage we designed rHAMs vector that can change its nucleic acid sequence at the binding site of miR171a to HAMs,but does not change the protein sequence by using codon degeneracy gained point mutant T0 generation seedlings by the transformation of rice.The homozygous lines were screened by antibiotics,and we found that rHAMs with over-expressed point mutation was more drought-tolerant than the wild type,and had a later heading and flowering stage than the wild type.