Analysis Of The Effects Of Different Cas9 Promoters On The Efficiency Of Soybean CRISPR/Cas9 Syste

Soybean genetic transformation is carried out by Agrobacterium tumefaciens mediated soybean cotyledon node method at present.However,the selection of transgenic recipients is greatly limited by soybean varieties,resulting in low efficiency of soybean genetic transformation.Therefore,improving the editing efficiency of CRISPR/Cas9 can improve the efficiency of positive and edited seedlings in transgenic materials at very low transformation efficiency,and accelerate the acquisition of mutant genetic materials,This has played a promoting role in the study of soybean gene function.CRISPR/Cas9 system,as an efficient genome editing system,has been widely used in animals and plants.Several Cas9 gene promoters,such as RPS5A and YAO,have been reported to improve the genome editing efficiency of CRISPR/Cas9 system.The effect of different Cas9 promoters in soybean on the efficiency of CRISPR/Cas9 genome editing system has not been clarified.This study selected six highly efficient Cas9 promoters with known functions（including p35S,pGmRPS5Ab,pGmRPS5Ac,pAtRPS5A,pGmYAO,pZmUbiquitin）and one promoter with unknown endogenous functions in soybean（pGmHE）to construct CRISPR/Cas9 gene knockout vectors.The editing efficiency of these Cas9 promoters on soybean endogenous genes GmSPA1a and GmEID1 was tested through the Agrobacterium mediated soybean rooting system.The main results are as follows:1.Screening high efficient Cas9 promoters in soybean,which the relative expression of 11genes in different tissues at the same time was compared to find a higher expression.It was found that Glyma.19G29210 was higher than other 10 genes in root,stem,compound leaf,single leaf and apical meristem.It can be used as a high expression gene for subsequent experiments and named High Expression gene（GmHE）.2.Screening for efficient endogenous Cas9 promoters in soybeans,which RPS5A in Arabidopsis was used as a reference gene sequence and seven members homologous to RPS5A（AT3G11940）were identified.Analyzing the gene structure of GmRPS5A members from the perspectives of evolution,motif,and gene structure,it was found that RPS5A family members have relatively conservative domains that can be used as target genes for further analysis.3.By analyzing the editing efficiency of soybean endogenous promoters on target genes,pGmRPS5Ab has the highest editing efficiency,with an average of over 60%of edited roots.However,pAtRPS5A,p35S,and pZmUbiquitin have higher editing efficiency on downstream genes than pGmYAO and pGmRPS5Ac.4.On the analysis of editing preferences of different Cas9 promoters for target genes,the results showed that pGmRPS5Ab and pAtRPS5A had low selectivity for downstream target genes,high editing efficiency for different genes,and remained stable.5.The analysis results of the target site sequencing peak plot show that in the sequencing peak plots using the pGmRPS5Ab and pAtRPS5A promoters,the peak proportion is relatively high,with 64%and 58.6%,respectively;In the sequencing peak plot using p35S,the proportion of low peaks is relatively high,at 63.3%.This indicates that the pGmRP5SAb and pAtRPS5A promoters not only have high editing efficiency,but also have good editing effects,which are more conducive to isolating homozygous mutant plants in the next generation.In this study,the CRISPR/Cas9 system was used to determine the editing effect of soybean hairy roots.By observing the status of heterozygous mutant sequencing peaks in the hairy roots,the editing effect was qualitatively determined,providing a reference for the probability of obtaining homozygous mutants in the T₁ generation.So far,there have been no similar reports,but the determination of the peaks and low peaks in the sequencing peak plot is related to the number of mutant DNA molecules in the sequencing product.That is,the more the target site DNA content is,the higher the sequencing peak plot becomes,and the probability of isolating mutants in the next generation is relatively higher.This provides theoretical support for this method.After obtaining stable soybean transgenic plants,the accuracy of this new method will be accurately determined by calculating the probability of obtaining homozygous mutants from T₀ to T₁ generations.