The Development And Application Of Egg-cell Specific Expression CRISPR/Cas9 Genome Editing System In Soybean

Soybean(Glycine max(L.).Merr.)is one of the important grain and oil crops in the world.Soybean is a paleotetraploid-evolved diploid plant.Soybean genome is large and highly repetitive.The whole genomes of some varieties and resources of soybean have been sequenced,which lay a foundation for the study of soybean gene functions.However,due to the recalcitrance and low efficiency of stable transformation of soybean,and impact of different varieties in transformation,the research of soybean gene functions is lagged.Because of the cost-effectiveness,high efficiency and easy operation,CRIPSR/Cas9 genome editing technology has been widely used in the research of almost of all the organisms,especially in crop improvement,gene functions and breeding.At present,CRISPR/Cas9 has successfully induced mutations of targeted soybean genes.However,due to the existence of chimeric in transformation,the mutations induced by constitutive promoters are non-inheritable.In this study,we mainly focused on the new specific expression soybean CRISPR/Cas9 genome editing system,editing efficiency and simultaneous editing of multiple target sites.The major results were obtained as follows:1.Different CRISPR/Cas9 systems were constructed for transgenic hairy roots of soybean,and the single target site and double target sites mutations of soybean endogenous genes were successfully induced.Among them,the 2X35Sp-driven CRISPR/Cas9 genome editing system could efficiently induce double target sites mutations,and the mutation efficiency was related to the activity of guide RNA.2.On the basis of the successful editing of endogenous genes in transgenic soybean hairy roots constitutively expressed with CRISPR/Cas9,a simple and convenient CRISPR/Cas9 system was designed and constructed for multi-target sites editing.Visual GFP tag and plant herbicide Bar tag were used to screen positive transgenic plants or non-transgenic mutants.It could be used for simultaneous editing of 2 or 4 or 6 target sites in soybean hairy roots or transgenic plants.3.For the specifically expressed CRISPR/Cas9 system,four different egg cell promoters(two from Arabidopsis thaliana: At P5 p,At EC1.2e1.1p;and the other two from soybean: Gm EC1.1p,Gm EC1.2p)were used to express Cas9 in egg cell and at the early stage of embryo stage.Then,the Agrobacterium tumefaciens-mediated stable transformation of soybean was performed.Among them,At EC1.2e1.1p:Cas9 successfully induced heritable mutations of Gm AGO7 a in T1 generation,the mutation efficiency was 26.8%.Both Gm AGO7 a and Gm AGO7 b were detected in T2 generation transgenic plants,indicating that At EC1.2e1.1p:Cas9 continued to induce target sites editing in T2 generation egg-cell and in early embryo development.The mutation efficiency of Gm AGO7 b is very low,which is consistent with the results of transgenic hairy roots,indicating that using hairy root system to detect the activity of guide RNA is an effective method to ensure high mutation efficiency.4.Arabidopsis thaliana was assistantly used to study the editing efficiency of four different egg cell-specific CRISPR/Cas9 systems.The results showed that in T1 generation,At EC1.2e1.1p:Cas9induced biallelic mutations in At RPS4,and the mutations were heritable.In T2 generation,At P5 p,At EC1.2e1.1p and Gm EC1.1p all produced a collection of mutations at the At RPS4 targets with frequencies ranging from 8.3% to 42.9%,but only At EC1.2e1.1p:Cas9 system could induce the At RPS4 B editing successfully.No mutations were detected at each target by Gm EC1.2p:Cas9.Through genetic segregation of plants,non-transgenic single-target and double-targets homozygous mutants were obtained in T3 generation.These results also proved that the editing efficiency of target sites was closely related to the activity of guide RNA.Phenotype identification also showed that the mutants based on WS background were effective.Put together,our study established the CRISPR gene-editing platform to generate inheritable mutants of soybean and Arabidopsis without the complication of somatic mutagenesis with the support for the study of functions of soybean and Arabidopsis genes.