Construction And Functional Evaluation Of Bivalent CRISPR Vector Of FAD2 Gene Family

Soybean is one of the most important grain and oil crops in our country and even throughout the world.In the daily diet,soybean oil supplements a large number of essential fatty acids for human body,which is rich in unsaturated fatty acid and easy to be oxidized.As a monounsaturated fatty acid,oleic acid is one of the fatty acids with the highest nutritional value and an important component of soybean oil.It possesses the characteristics of stability and difficulty to be oxidized,and plays an important physiological role in human body,being able to prevent cardiovascular disease,and reduce low density lipoprotein and blood viscosity.Although linoleic acid,gamma linolenic acid and other polyunsaturated fatty acids are beneficial to human body,they are not easy to store and more incline to spoil.Therefore,increasing the content of oleic acid and reducing the content of linoleic acid,gamma linolenic acid and other polyunsaturated fatty acids is one of the important breeding objectives for genetic breeders to breed new soybean varieties.In the synthetic pathway of fatty acid,FAD2 family gene are the key gene to regulate the generation of linoleic acid from soybean oleic acid.It is reported that the researchers have used RNAi technology,antisense RNA technology,artificial nuclease technology,etc.to regulate the expression of FAD2 family gene,and obtained high oleic acid mutant materials.But,the reports about the direct use of CRISPR/Cas9 technology to study the function of FAD2 family gene are rare.CRISPR/Cas9 technology,as a new gene editing method,is widely used in the research of gene expression regulation due to its characteristics of simplicity,easy to operate,time-saving,labor-saving,etc.,which provides technical support for improving the content of soybean oleic acid.In this study,two genes,Gm FAD2-2A and Gm FAD2-3,were selected as the main target genes.CRISPR/Cas9 gene editing technology was used to edit the target gene in a fixed-point manner and construct a bivalent CRISPR/Cas9 expression vector.Genetic transformation was carried out in the receptor Jinong 38,and molecular biological detection and investigation of agronomic characters were conducted after obtaining a positive strain.The aim is to elucidate the functions of soybean Gm FAD2-2A and Gm FAD2-3 in soybean.To investigate whether blocking the expression of FAD2 family genes can achieve the purpose of increasing soybean oleic acid and reducing linoleic acid.The main test results are as follows:1.Aiming at gene Gm FAD2-2A and Gm FAD2-3,two pairs of g RNA were designed at different exons respectively.And two bivalent CRISPR/Cas9 expression vectors（hereinafter referred to as p Cas9-FAD2-2A-3）of Gm FAD2-2A and Gm FAD2-3were constructed by the means of CRISPR/Cas9 vector construction kit.The homology comparison between sequencing results of the company and the target gene sequence by DNAMAN software was conducted,and the results were basically consistent,reaching more than 99%.2.The p Cas9-FAD2-2A-3 vector was introduced into soybean material Jinong 38by Agrobacterium-mediated method.Through PCR amplification,the screening marker genes Bar and Cas9 protein genes on the vector were detected.The results showed that all the vectors were successfully transferred into the receptor material.3.The sequencing analysis of the T₁ generation materials containing p Cas9-FAD2-2A-3 vector was carried out,and the editing sites of CRISPR/Cas9system were detected.The results showed that there were three plants editing at two target spots in the progeny of transformed plants,and the editing mainly included the deletion,insertion and replacement of bases.4.The integration effect was detected by the means of Southern blot hybridization technique.The results showed that the target fragment was integrated into soybean genome in the form of dual copy or single copy,and the plants with stable hereditary characters were obtained by the multiple generations.The relative expression quantity of genetic transformation RNA（m RNA）of Gm FAD2-2A and Gm FAD2-3 was detected by fluorescence quantitative PCR.The relative expression quantity of the two genes was significantly lower than that of the control group.5.The analysis result of the fatty acid composition of transformation plant of mutant material showed that oleic acid of the mutant material containing p Cas9-FAD2-2A-3 was 50%higher than that in the control group.Therefore,it was speculated that Gm FAD2-2A and Gm FAD2-3 genes in soybean had the regulatory effect on fatty acid metabolism,and the efficiency was higher when the two genes interacted.6.The results of agronomic traits analysis of the offspring after transformation showed that there were no significant differences in plant height,number of main stem nodes,branching number of single plant,leaf shape,flower color,middle skin color,hilum color,growing stage and other agronomic traits.