| Part 1 Targeted Gene Editing in Soybean by TALENs and CRISPR/Cas9Soybean[Glycine Max（L.）Merr.],originated in China,is one of the most important oil and high-protein crops,containing a variety of physiologically active substances that are beneficial to humans,such as isoflavones,etc.In addition,soybean is an important soil improving crop in the agricultural production system（capable of nitrogen fixation and less demand for fertilizer）.Targeted genome editing technologies（also named gene targeting technologies）operate on specific loci of the genome,followed by changes in the DNA sequence of the site,such as indel,insertion or substitution of genes,or deletion of large fragment.Currently widely used is TALENs（transcription activator-like effectors nucleases）and Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated（Cas）9 technologies.Targeted genome editing technologies rely on transgenic technology,but after gene targeting the crops do not contain any newly introduced genetic material or exogenous DNA,mean that gene targeting is a "non-transgenic genetic modification".Compared with the low efficiency of traditional breeding,and the biosafety of foreign DNA in transgenic breeding technology,Targeted genome editing technologies are superior to the former in breeding efficiency and controllability.Here we compared the TALENs and CRISPR/Cas9 technologies for targeted genome editing in soybean.The main results were listed as followed.1.Application of TALENs technology in soybean genome editing.GmPDS11（Glyma.11G253000）and GmPDS18（Glyma.18G003900）were selected as the target genes,two types of gene targeting were designed:D1 simultaneously targets two genes;S1 and S2 specific target GmPDS11 Hairy-root transformation was used to assess the efficiency of targeted mutagenesis.The results showed that the efficiency of single site mutations was 17.5-21.1%,and the efficiency of mutations at both targets was 6.25%.The type of mutations is mainly the base（1～30 bp）deletion,less base insertion.TALENs preferentially bind to the target site of the "0" position for T base（the”0”position of naturally TALE’ target is almost all T）,but this preference is not strictly,as they can also bind to the target site when the "0" position is C base.2.Application of CRISPR/Cas9 technology in soybean genome editing.GmPDS11 and GmPDS18 were selected as the target genes,two types of gene targeting were designed:D7 simultaneously targets two genes;S11 and S12 specific target GmPDS11,S13 specific targets GmPDS18.Hairy-root transformation was used to assess the efficiency of targeted mutagenesis.When the CRISPR/Cas9 system used the Arabidopsis U6-26 promoter,the efficiency of mutations at single site was 11.7-18.1%,and the efficiency of D7 targeting both loci was 12.5%.Mutation type is mainly indels（1～38bp）.Eight U3 and eleven U6 genes were identified in the soybean genome.When the CRISPR/Cas9 system used the GmU6-16g-1 promoter,the efficiency of D7 was 43.4%（GmU6-16g-1 promoter length 616 bp）and 48.1%（GmU6-16g-1 promoter length 350 bp）,and all exhibited mutations at both targets.In addition,the D7-pSC-AtU6 vector was introduced into soybean cotyledon nodes using Agrobacterium tumefaciens,and albino adventitious buds（pds mutant phenotype）were observed during subculture stage.3.Comparison of TALENs and CRISPR/Cas9 technologies soybean genome editing.Firstly the assembly process of TALENs is time-consuming and laborious,relying on a large amount of sequencing,while the construction of Cas9 vector is relatively simple,only need simple enzymatic digestion and connection.Second,the mutation efficiency of TALENs was slightly higher than the CRISPR/Cas9 using the AtU6-26 promoter but much lower than that of CRISPR/Cas9 using the GmU6-16g-1 promoter in hairy roots.CRISPR/Cas9 is more suitable for simultaneous editing of multiple homoeoalleles in hairy roots,and GmU6-16g-1 is a suitable U6 promoter for the expression of sgRNA in soybean.Part 2 Map-based Clone and Functional Analysis of the Chlorophyll-deficient Gene cd1Plant leaf color is a comprehensive reflection of the pigments,under normal circumstances they show green as that chlorophylls accounted for the main component.Chlorophylls are indispensable element in photosynthesis,involved in the assembly of photosystem and absorption of light energy.Normal chlorophyll biosynthesis and degradation is of great signification to all photosynthetic microbes and green plants.Chlorophyll-deficient mutants are ideal materials for studying the mechanism of chlorophyll metabolism,biogenesis and development of chloroplasts,signal transmission between chloroplasts and nuclear,photosystem,etc.In this study,a previous isolated soybean chlorophyll-deficient mutant（named cdl,isolated from an EMS-induced mutant bank of cultivar Nannong 86-4）was used,the cdl gene was fine-mapping and functional analysis.The main results were listed as followed.1.Genetic analysis indicated that the mutation was controlled by a recessive allele of nuclear gene.To map the cdl locus,a F2 mapping population was generated by crossing the cdl as female parent with Williams 82 as male parent.Bulked segregant analysis（BSA）and fine-mapping revealed that the cdl was located between locus to a 153 kb interval between SSR marker M1 and 150286 on chromosome 15.24 predicted genes were located in this region,and Glyma.15G080200（encoding the CHLI subunit of Mg-chelase）was the most probable candidate gene.DNA sequencing revealed that a single base change,G1709A,occurred in the third exon of Glyma.15G080200,causing a missense mutation（D278N）in the cdl.The GmCHLI1b was knocked out by using CRISPR/Cas9 technology,and adventitious buds were observed in the subculture stage.RCR/sequencing was performed after DNA extraction and found that the GmCHLI1b gene was mutated.2.There are four GmCHLI copies in the soybean genome,hereafter as GmCHLI1a（Glyma.13G232500）,GmCHLI1b（cdl）,GmCHLI2a（Glyma.07G204300）,GmCHLI2b（Glyma.13G171800）.GmCHLla/b are the major isoforms of CHLI in soybean.Compared with GmCHLI1b,the ATPase activity of Gmcdl was significantly decreased.In addition,the yeast two-hybrid experiment showed that the mutation site D278N weakened the interaction between Gmcdl and other CHLI subunits,but did not affect its interaction with CHLD subunit.3 The globle expression level of related genes in chlorophyll synthesis pathway was analyzed by using the transcript data at four time points（V3）:dawn,mid-day,dusk and midnight.Compared with Nanong 86-4,the lower chlorophyll content of the mutant cdl at dawn resulted in the up-regulation of genes in chlorophyll synthesis pathway.With the passage of time,the lower activity of Mg-chelate in the cdl hindered the normal synthesis rate of chlorophyll,and the feedback regulation lead to the down-regulation of most genes.In addition,the activities of SOD,APX and POD in the leaves of mutant cdl were significantly increased,suggesting that the ROS level in the mutant was higher than that in the Nannong 86-4,indicating that the redox balance of the leaves was broken.4.In the yeast two-hybrid system,soybean thioredoxin GmTrxF1/2（Glyma.09G249200/Glyma.18G243200）and NADPH-dependent thioredoxin reductase C GmNTRC1/2（Glyma.02G185300/Glyma.10G113100）were not interact with GmCHLI1b.In addition,bimolecular fluorescence complementary（BiFC）experiments also showed that GmTrxF1/2 and GmNTRC1/2 did not interact with GmCHLI1b.These results suggest that GmTrxF1/2 and GmNTRC1/2 may not directly participate in the redox regulation of GmCHLI1b. |
