Analysis Of Soybean U6 Promoters Activity And Its Application In Gene Editing

Soybean is the fourth largest crop in the world,an important economic crop,and plays an important role in agriculture.Soybean is the crop with the highest protein content in crops,with a protein content of 35%-40%.Soybean is a high-quality edible oil with an oil yield of 18%-20%.It is an important source of human protein and edible oil.In addition,soybean also can be used as feed,raw materials of industrial products;Soybean seeds can also be processed into a variety of food products,such as protein products for flushing and regulating drinking protein and additives.The continued increase in demand for soybean production has forced the need for high-quality,high-yield,and highly resistant soybean varieties.With CRISPR-Cas9,the improvement of soybean varieties can be achieved quickly and efficiently.In the CRISPR-Cas9 system,the U6 promoters are often used to drive the transcription of sgRNA,which is an important element in the CRISPR-Cas9 system.The soybean U6 promoter for soybeans and capable of efficient transcription has not been reported,and the U6 promoter between species with distant genetic relationships may not be suitable.The purpose of this study was to screen a soybean U6 promoter with higher transcriptional activity and editing efficiency.Methods: 11 U6 soybean promoters were cloned,and the transcriptional activity of soybean U6 promoters in soybean hair roots and Arabidopsis thaliana through was analyzed by driving the expression of the GUS gene.At the same time,four soybean genes were selected as the target genes for gene editing to induce soybean hair roots formation.The mutation efficiency of soybean target gene was detected by DNA polymerase chain reaction and restriction endonuclease digestion(RE-PCR).The promoters with the most efficient editing efficiency were screened out,can be used to knock-out or silence soy-related functional genes to obtain nutrient-rich and high quality soybean,which provided the basis for the application of CRISPR-Cas9 system in soybean molecular breeding.The main results are as follows:1.Eleven U6 promoter were cloned at the whole genome level of soybean with fragment size of 352 bp,256 bp,342 bp,236 bp,342 bp,257 bp,279 bp,314 bp,280 bp,306 bp,294 bp.Based on 11 GmU6 promoters,p3301-GUS-GmU6-1~p3301-GUS-GmU6-11 eukaryotic expression vectors were constructed and used for plant transformation.2.In this study,the expression vectors transferred into Agrobacterium rhizogenesK599 and Agrobacterium rhizogenes GV3101 by liquid nitrogen freeze-thaw method,respectively,which were used to induce soybean hair roots and transform Arabidopsis thaliana.The transcriptional activity of 11 GmU6 promoters were systematically analyzed and compared.As a result,we found that the transcription levels of the GmU6-1,GmU6-4,GmU6-7,GmU6-8,GmU6-10 and GmU6-11 promoters were significantly higher in the hairy roots of soybean than in the GmU6-5 promoter.The transcriptional activities of the GmU6-2,GmU6-3,GmU6-4,GmU6-7,GmU6-8,GmU6-10 and GmU6-11 promoters in Arabidopsis thaliana were higher than those of GmU6 promoter.Based on the relative expression levels of 11 U6 promoters in the hairy roots and Arabidopsis thaliana leaves,GmU6-4,GmU6-7,GmU6-8,GmU6-10 and GmU6-11 promoters had relatively high transcriptional activity.3.In this study,we selected four soybean genes as the target genes for gene editing,and designed corresponding sgRNAs for each gene.Finally,Glyma06g14180-pCas9-GmU6-1~ Glyma06g14180-pCas9-GmU6-11,Glyma03g36470-pCas9-GmU6-1~ Glyma03g36470-pCas9-GmU6-11,Glyma11g253000-pCas9-GmU6-1~ Glyma11g253000-pCas9-GmU6-11,Glyma14g04180-pCas9-GmU6-1~ Glyma14g04180-pCas9-GmU6-11,a total of 44 vectors,were used to transform soybean hairy roots.4.The PCR-RE method was used to detect mutations in soybean hairy roots transformed by 44 vectors.It was found that the mutation types of 11 GmU6 promoters in different genes were different.The types of mutations involved in Glyma03g36470,Glyma14g04180,and Glyma11g253000 were mainly a base deletion,and most of the mutation types of Glyma06g14180 were base insertions and deletions.The editing efficiency of the GmU6-8 and GmU6-10 promoters in the three genes Glyma06g14180,Glyma03g36470,and Glyma14g04180 were 22.1%,26.6%,and12.1%;20.5%,14.7%,and 15.9%.Combining the transcriptional activity of 11 GmU6 promoters in soybean hairy roots and Arabidopsis thaliana,we found that GmU6-8and GmU6-10 promoters have relatively high gene editing efficiency in the CRISPR-Cas9 system(average mutation rates are 20.3% and 20.6%).