Research On The Creation Of Low-cadmium Mutants In Rice Using CRISPR/Cas9 Technology

Rice is an important food crop in China.With the rapid development of social industrialization,the problem of excessive cadmium in rice caused by environmental pollution in China is becoming more and more serious.Therefore,it is urgent to reduce cadmium pollution in rice and cultivate rice varieties with low cadmium accumulation.CRISPR/Cas9 technology is a rapidly developing gene editing technology in recent years,which can effectively and directionally improve biological quality traits.In this study,the Os NRAMP5 gene(LOC＿Os07g15370)of Japonica Rice "Jiahua 1" was directionally edited by CRISPR/Cas9 technology.Through agrobacterium mediated transformation and rice genetic transformation process,combined with DNA molecular identification,the low cadmium rice mutant lcd1,which was genetically stable and did not contain transgenic components,was obtained,which verified that the low cadmium rice material could be quickly obtained by CRISPR/Cas9 technology.Through the analysis of physiological and agronomic characters,transcription level and cadmium content in various tissues of lcd1,the following research results were obtained:(1)The knockout expression vector was constructed based on the principle of CRISPR/Cas9 technology,and the genetically stable rice low cadmium mutant lcd1 without transgenic components was successfully obtained by Agrobacterium mediated transformation;(2)The results showed that with the increase of cadmium concentration,the germination potential and germination rate of lcd1 were slightly better than that of wildtype "Jiahua 1",and there was no significant difference between wild-type and lcd1;(3)Tissue specific expression analysis of rice showed that Os NRAMP5 gene was constitutively expressed and expressed most in roots,followed by panicles;The results showed that the mutation site of lcd1 might mainly affect the absorption and accumulation of cadmium in rice before and after heading;(4)The determination of cadmium content in brown rice of low cadmium mutant lcd1 under different cadmium treatment conditions showed that the cadmium content in brown rice of lcd1 decreased more significantly in low and medium concentration cadmium polluted soil,indicating that lcd1 rice mutant material might be more suitable for cultivation under medium and low cadmium pollution conditions;In addition,there were differences in cadmium absorption and accumulation of lcd1 mutant under field water management.