Preliminary Function Study Of GmPT1 And GmACP1 Using CRISPR/Cas9 Technology

Low phosphorus(P)stress is a key factor limiting plant growth and production,which is common in most basic agricultural soils.Most of the soils are rapidly immobilized by applying phosphate,and most of the phosphate fertilizer applied each year is fixed by adsorption,sedimentation and transformation.However,excessive phosphorus input can cause eutrophication.For plants,crop genetic engineering is the most effective way to deal with the mechanism of low phosphorus stress and improve the efficiency of phosphorus.In order to cope with the deficiency of phosphorus and the maintenance of phosphate homeostasis,the plants themselves have different adaptive mechanisms,including changes in root morphology,inducted of inorganic phosphorus and acid phosphatase,and inducted of phosphate transporters.There are two ways to improve the phosphorus nutrition of crops.One is to improve the effectiveness of phosphorus in soil by reasonable fertilization,so that the use of phosphorus.Two is the screening and cultivation of high efficiency can make full use of soil phosphorus and low phosphorus crop varieties.However,the screening of the high performance low phosphorus soybean varieties less,which makes the traditional cross breeding limited.Moreover,the traditional breeding process is tedious,the breeding cycle is long,and the traits of the offspring are less predictable.At present,with the rapid development of genetic engineering technology,transgenic breeding has become an important method for breeding researchers to obtain excellent crop varieties.In this paper,we used the technology of CRISPR/Cas9,soybean low phosphorus tolerance related genes GmPTl and GmACPl were edited by designing different U6 promoter and Cas9 promoter.Transgenic plants were obtained by Agrobacterium mediated Soybean Cotyledonary Node Method for genetic transformation,for the further study of soybean Low-P tolerance mechanism.The main results of this paper are as follows:1.We used the original vector(with Arabidopsis thaliana U6 promoter and Cas9 protein promoter)and two other vectors,modified by Dr.Du Hongyang,to determine the mutation efficiency of the rooting transformation of soybean Jake varieties.The results showed that the root appearance.The mutation rate of the vector with Arabidopsis thaliana U6 promoter was about 20%,while the mutation rate of the vector with soybean U6 promoter was about 30%.The mutation rate of the vector with maize Ubi promoter was about 30%,while the mutation rate of the vector with soybean Ubi promoter was about 50%,which indicated that the promoter of soybean was more likely to initiate the expression of gRNA and Cas9 protein in soybean,so that the directed mutations of the related gene could be easier to complete.2.This research used eight vectors with different genes and promoters,and detected 13 transgenic plants,the transformation rate is about 2.13%.3.Some of the T1 plants were planted and the leaves RNA was used to do qPCR.The results show that transgenic plants appear serious phenomenon of separation in T1,and the mutation efficiency is lower than that in the root hair transformation system.