Application Of CRISPR/Cas9 System In FaSGR Gene Knock-Out Of Tall Fescue

Tall fescue,as the perennial gramineous plant belonging to the Festuca is widely used in soil and water conservation and lawn greening because of its strong resistance,resistance to trampling and resistance to extensive management.But the cold-season turfgrass in the summer have "sleep" phenomenon,which limits the use of tall fescue in the summer,so the extension of the tall fescue green period is one of the important breeding objective.CRISPR/Cas9 system,as a new gene editing technology in recent years,has become the hot research area because of its advantages such as simple design principles,high precision,possibility to be edit multiple sites at the same time and the low cost.In this study,Ca(Cl O)2 instead of Hg Cl2 was used as a disinfectant to establish an efficient regeneration system;the Fa SGR gene knock out vector was constructed by CRISPR/Cas9 gene editing technique;agrobacterium tumefaciens-mediated vector into the callus of tall fescue for genetic transformation,and mutations and expression of SGR gene in transgenic plants were tested by sequencing and real-time PCR.This paper aims to lay the experimental foundation for the application of CRISPR/Cas9 system in the genetic improvement of tall fescue.The main results were as follows:1)Use Ca(Cl O)2 as a disinfectant and three different pre-treatment methods with different concentrations of Ca(Cl O)2 solution and different disinfection time to disinfect tall fescue seeds,with Hg Cl2 as controls.The results of callus culture showed that that infection rate of Only Ca(Cl O)2 was significantly higher than other treatment;Ca(Cl O)2 in high concentration treatment has lower callus rate than that in low Ca(Cl O)2 concentration;the rate of callus and total callus in Ca(Cl O)2 treatment was higher than Hg Cl2 treament.The best disinfectant combination is stirred in a sterile environment with Ca(Cl O)2+3%Ca(Cl O)2 Soak for 60 min.2)Primers were designed for the first two exons of the tall fescue SGR gene(Gene bank Accession NO.319430080)CDS region and their base sequences were determined;the CRISPR/Cas9 target design principle was used to design Target design of the obtained exon base sequence.The DNA of the g RNA target was used to cleave the target detection kit and to detect the activity of the target g RNA which can reduce the target;finally target will be built to the VK005-05 vector and designated as Fa SGR-Cas9 vector,which carries Kan and Hyg resistance.3)The vector Fa SGR-Cas9 was transferred into agrobacterium by freeze-thawing method and cultured on LB solid medium containing different Kan concentrations.Finally,the optimal concentration of Kan antibiotic on Agrobacterium grown on LB solid medium was 75 mg·L-1,and the optimal concentration of Hyg was 75 mg·L-1 for the first round of screening,100 mg·L-1 for the second.The resistance callus rate was 6.7%,the resistance callus differentiation rate was 0.5%,and the ratio of phenotype to normal phenotype was about 2:1.4)In order to detect whether the transgenic plants were positive plants,the total DNA of transgenic plants of 7 normal phenotypic transgenic plants and 3 whitening phenotypes were extracted for PCR-positive agrobacterium tumefaciens as positive control,Wild type plants and water as negative control;The results of PCR showed that only one of sample has no target bands in the normal phenotype,and other samples contained the target bands,Nine samples of the target bands were sequenced and the results showed that although the plasmid was successfully transferred into the plant,the target sequence base didn't change.5)In order to verify the induction effect of SGR gene on chlorophyll biodegradation of common wild type plants under dark conditions,and to compare with the transgenic tall fescue plants,the wild leaves of tall fescue were selected for in vitro induction,then genes were extracted from leaves darkness with 0,3,6,9 d and were transfected into c DNA for real-time PCR.The results showed that the SGR gene of senescence leaves was the highest at 6 day.6)Although the target base of the plants were not change,in order to verify the final effect of the expression level of the transgenic plant on the SGR gene,the positive plant RNA was extracted and identified as c DNA then detection by q RT-PCR.the leaves of wild tall fescue were used as control;the results show that the SGR gene in transgenic plants showed a downward trend,indicating that although the target base did not change,CRISPR/Cas9 system still inhibited the expression of SGR gene.