Preliminary Construction Of Soybean 'Tianlong 1' Mutant Library And CRISPR/Cas9 Technology Platform

Soybean [Glycine max(L.)Merr.] belongs to Glycine genus,Faboideae subfamily,Leguminosae family.It is one of the most important economic crops planted worldwide.It is the main source of plant protein and also one of the vegetable oil.Since soybean is an ancient tetraploid,about 75% of the genes are expressed in the form of homologous genes,and its genomic structure and low genetic transformation efficiency limit the development of functional genomic research.With the initial completion of the soybean genome sequencing,soybean functional genomics research has gradually become the focus of research.Mutants are important materials for gene function research and are the basic materials for soybean genetic breeding.Therefore,this study used 60Co? ray and ethyl methanesulfonate to treat soybean "Tianlong No.1" to construct a soybean mutant library and edit the soybean "Tianlong No.1" flower development related genes Gm PTL and Gm AP1 by constructing the CRISPR/Cas9 technology platform in order to obtain mutants related to soybean flower development.The specific experimental results are as follows:1.Construction of Soybean Mutant LibraryThe phenotypic traits of the mutants of the M2 and M3 generations of the soybean "Tianlong No.1" seeds were investigated and statistically analyzed,and 140 mutants were obtained through further screening and analysis,including leaf,flower,stem,pod and other traits.Among them,63 for leaf variation materials,75 for plant type variation materials,11 for flower variation materials,18 for pod variation materials,50 for seed variation materials and 28 for mature variation materials were obtained.Different variant traits are superimposed in the same line.2.Molecular marker identificationFrom the soybean genome database,60 pairs of soybean SSR molecular marker primers similar to the average distribution on 20 pairs of chromosomes were selected to analyze 138 experimental materials.26 pairs of primers were able to amplify the differential bands,and a total of 105 materials were The presence of SSR variability was detected and it was demonstrated at the molecular level that these materials were mutated.3.Construction of the CRISPR/Cas9 technology platform(1)Based on the protein sequence of the ptl and AP1 genes of Arabidopsis thaliana,the soybean database was compared in the soybase.org database,and the highest homology was named Gm PTL and Gm AP1.By q PCR detection,it was found that Gm PTL and Gm AP1 were specifically expressed in soybean flowers and sepals relative to leaves.Three CRISPR/Cas9 targeting sites were designed on their exons: Gm PTL-1,Gm PTL-2 and Gm AP1-1.(2)The Gm PTL and Gm AP1 knockout vectors p HEE401E-Gm PTL-1,p HEE401E-Gm PTL-2 and p HEE401E-Gm AP1-1 were constructed and transformed into Agrobacterium K599,and transformed into soybeans by Agrobacterium-mediated method.Cotyledons,demonstrating that the constructed vector is capable of enabling site-directed editing of genes in soybean hairy roots.(3)Agrobacterium tumefaciens-mediated transformation of soybean cotyledonary node system was optimized for Agrobacterium strains and hormone concentrations.Agrobacterium LBA4404 showed better and more stable transformation efficiency.The vectors p HEE401E-Gm PTL-1,p HEE401E-Gm PTL-2 and p HEE401E-Gm AP1-1 were transformed into Agrobacterium LBA4404,and the soybean genome was edited by the optimized Agrobacterium tumefaciens-mediated cotyledonary node transformation system to obtain T1 and a total of 7 plants were obtained.(4)The T1 generation strain DNA was amplified by specific primers,and it was found that a total of 6 soybean genes were edited,namely T2000-1,T2000-1a,T2000-3,T2000-4,T2000-5,T2000-6.