Using CRISPR/Cas9 Technology To Create A Mutant Library Of Highly Expressed Genes In Soybean Seeds

Soybean[Glycine Max（L.）Merr.]is an important legume crop being rich source of edible seed protein and oil content.Although,its primary center of origin is China,but is now cultivated globally as an important cash crop.Soybean is a paleotetraploid crop,and75%of its genes exist in multiple copies in the soybean genome.Complex genome structure of the soybean has substantially reduced the gene transformation success and efficiency in this crop.This in turn has limited the functional genomics research in soybean as well as its improvement.In this regard,the mutants have emerged as an important resource for the verification of the gene function in soybean.Recent advances in the genome sequencing have allowed to detect the mutants generated via spontaneous mutation,but these mutations are not enough to fulfill the needs of soybean improvement.Hence,an immediate need has been felt by the researchers to create an artificial mutant library by using the approaches of reverse genetics for the high-throughput and large-scale mutant identification.In theory,often we suppose to end up with mutants for all different genes of the crop genome.In this study,the soybean seed-specific gene mutant library was created by CRISPR/Cas9 technology,and were used to obtain mutant plants and T₀ generation plants by Agrobacterium-mediated cotyledonary node method,and the gene number of the identified plants was determined at the same time.The main results of the present study include:1.A total of 1193 sg RNA were designed by selecting 481 highly expressed genes related to soybean yield and quality.Sanger sequencing of 20 random Escherichia coli monoclones showed that sg RNA accuracy was up to 95%.It can be converted directly from soybeans.2.The soybean transformation efficiency is affected by a variety of factors/parameters.However,among the different parameters affecting the soybean transformation efficiency,such as varieties,co-culture time and screening agents,etc.This experiment changed the length of the co-cultivation time,the types of antibiotics and their doses,and the screening doses in the soybean transformation process,so as to optimize the transformation steps and related parameter values that are more suitable for the mutant library to obtain mutant plants.3.The CRISPR/Cas9 technology was used to construct a sg RNA library of high-expressed genes in soybean seeds for genetic transformation to obtain mutant plants.In this study,the T₀ generation plants with positive targeting were obtained and T₁ seeds were preserved,and the targeting genes were determined.The target gene of the mutant plant is GmMetE1,which is a highly expressed gene in the seed.Cloning and preliminary bioinformatics analysis of this gene were carried out.