Genomic Editing Of Cis-regulatory Sequences Of Rice Blast Resistance-related Gene OsBR1

Rice?Oryza Sativa?is the largest grain crop and plays an important role in national life and national food production security in China.There are many reasons for affecting high yield and stable yield of rice,among which the impact of pests and diseases is one of the main reasons.Among them,rice blast is the first of the three major diseases of rice.However,the physiological races of Magnaporthe oryzae mutate rapidly,and with the changes of time,nature and cultivation conditions,the resistant varieties are no longer resistant to new dominant physiological races or the resistance is weakened.Therefore,making full use of basic resistance gene resources and cultivating varieties with good basic resistance has become a consensus for improving the long-term disease resistance of rice^[1].In recent years,our laboratory has carried out some research on rice blast resistance related genes,created a large number of RNAi materials and overexpression materials,and screened some negatively regulated rice disease resistance-related genes.The OsBR1?rice blast resistance gene 1?gene that is quantitative trait resistant to sheath blight.This study used CRISPR/Cas9 technology to predict and analyze the cis-regulatory sequence of the promoter region of one of the selected disease-resistant genes OsBR1 and constructed a series of CRISPR/Cas9 vectors for gRNAs targeting multiple promoters.Furthermore,a large number of OsBR1 gene promoter mutant materials of pPLY17,pPLY46 and pPLY52 were screened by red fluorescent protein gene mCherry-assisted screening and PCR sequencing.In this study,a detailed gene editing analysis was performed on the T₀ and T₁generations of pPLY17 mutant material,and 21 T₁ generation T-DNA-free homozygous plants were identified and initially screened.The three target sites were mainly point mutations.And each target site mutation type is fixed in the same position,and the specificity is high;of course,the large fragment deletion mutant homozygous plant pPLY17-T0-20 is also screened.At the same time,we summarized and analyzed the mutation types of these materials and found that there are a total of 10 mutation types,combined with quantitative analysis and promoter cis-element analysis.Finally,we screened out some materials with significant differential expression of OsBR1.We not only screened T-DNA-free rice materials using gene editing technology but also screened marker-free plants using the dual-fluorescence Ac/Ds transposon system,with an efficiency of 31.0%.Finally,we also used Southern hybridization technology,the presence of GFP gene was not detected,indicating that these marker-free plants do not contain T-DNA related sequences,the screening efficiency is higher than the single fluorescence system,and no false positives are detected.In the present study,we verified that the maize Ac/Ds transposition system can be routinely used to produce marker-free transgenic plants in rice.In terms of transgenic safety,the Ac/Ds element is a native genomic sequence from maize,and the Ds-mediated transgene reintegration strategy requires a limited number of primary transformants and results in a complete transgene insertion with defined boundaries.Thus,the vector system can be used for marker-free transformation in rice and may be used for other crops,including refractory species in transformation.