Greating Low Cd-Accumulating Rice By CRISPR/Cas9 Technology

CRISPR/Cas9 is a highly efficient genome editing technology and is widely used in genetic improvement of rice and other crops by directional modification of genomic sequence to change gene function.At present,different degrees of cadmium pollution exists in part of farmland in China.The problem of“cadmium rice”has drawn public attention because of the excessove accumulation of cadmium in rice grains under cd-contaminated field conditions.Therefore,the most economical and effective way to solve the problem is to create rice varieties with low cadmium accumulation.In this study,the CRISPR/Cas9 system was used to edit the heavy metal transporter gene（OsNramp5）in two indica rice,chenghui 727（R727）and chuankang 606B（606B）,to develop new indica rice materials with low cd-accumulation and no transgenic components.The main research results of this paper are as follows:1.According to the target gene designed two target loci,editing vector was constructed by using CRISPR/Cas9,and the generation transgenic plants of R727 and606B were gained.In addition,the positive transgenic plants target was screened through the PCR analysis for hygromycin gene.2.Homozygous mutants without exogenous transformation elements were screened out by identifying the self-pollinated population of T₁generation.By sequencing analysis of edit loci and design of PCR amplification primers for detection,the R727 editing materials were divided into three genotypes,named A1,A2,A3.and the 606B editing materials were grouped into two genotypes,named B1 and B2.3.The relative expression levels of OsNramp5 gene in roots,stems,leaves and young ears（3-4 cm,8 cm）showed that the relative expression of target genes in editing materials was generally lower than that of receptor materials under the cd-contaminated soil.4.The cadmium content in brown rice of the T₂generation gene editing material under the cd-contaminated soil was analyzed,the mean cadmium content of brown rice in R727 editing material is about 0.12mg/kg,40%lower than that of wild type material.The average cadmium of brown rice in 606B editing material is about0.09mg/kg,69.5%lower than that of wild type material,All editong materials meet the national standard of less than 0.2mg/kg.5.The cadmium contenet of the seedlings in T₂generation gene editing materials under the hydroponic conditions of two cadmium concentrations（0μM and 7.8μM）was measured,Compared with wild parents,the cadmium content of the R727 editing material decreased by about 33%and the 606B editing material reduced by about25%,Moreover,the average plant height of the R727 editing materials was increased by 1.3 times,and the plant height of the 606B editing material was increased by 1.1times.