Cloning Of Tall Fescue FaSGR Gene And Construction Of Its RNAi Gene Silencing System

Tall fescue (Festuca arundinacea) the originated from Western Europe, North Africa, is turfgrass and forage widely used in temperate regions. It is widely applied in urban afforestation, soil and water conservation because it has strong resistance, traffic resistance, resistance to extensive management, long green period and better turf establishment. Common warm season turfgrassand cool-season turfgrass have seasonal dormancy, people prefer to select turfgrass with long green period. The summer dormancy phenomenon for Cool-season turfgrass is a major limited factor of the production and utilization, prelonging green stage is one of the main objectives of tall fescue breeding. In our study, the endogenous stay gene FaSGR of tall fescue was cloned, and the RNAi-FaSGR gene silencing vector system was constructed, and the vector was transformed into the tall fescue using Agrobacterium-tumefaciens mediated transformation in vivo in order to get tall fescue with prolonging green stage. Our study provide a experiment basis for RNAi silencing applying in the genetic improvement of turfgrass. Specific research methods and conclusions are as follows:1) The proline and MDA content of tall fescue leaves was the highest after 6 d dark treatment; its relative chlorophyll content gradually decreased, the proportion of yellow leaves area gradually increased along with the the prolongation of dark pretreatment time. Tall fescue leaves after 6 d dark treatment was used to extract RNA, the tall fescue FaSGR gene fragment (1346 bp) was successfully cloned.2) Bioinformatics analysis of tall fescue FaSGR gene fragment showed that the tall fescue FaSGR gene has about 99% homology with SGR reference sequence (GenBank Accession No.319 430 080). The analysis of structural properties of FaSGR gene fragments using bioinformatics analysis software or online websites showed that its protein molecular weight was 10.91 Kd, isoelectric point was 4.90; 32.13% a-helix,14.08% chain elongation and 53.79% random coil were predicted in representing protein secondary structure of FaSGR. The protein has not the transmembrane structure, and has 6 phosphorylation sites (>0.5) are the value number from 4 of 6phosphorylation sites were closed to 0.9. The protein fragment may be a non-secreted protein.3) FaSGR gene CDS center region was selected as a target site, primers (FAS-1 and FAS-2) of this region were designed. This fragment was cloned the fragment, tall fescue FaSGR gene RNAi expression vector pANDA-SGR with Ubiquitin promoter was successfully constructed through BP and LP connections using Gateway technology. The expression vector has the Kan and Hyg resistance.4) RNAi expression vector pANDA-SGR was transferred into Agrobacterium by electroporation method, its Kan antibiotic concentration in growth medium is 40 mg/L, the optimal concentration of the Rif antibiotic in the growth medium is 60 mg/L. The optimal screening approach for resistant callus was two cycle of screening using 50 mg/L hygromycin concentration. Rate of resistant calli obtained was 1.59%, and its resistant callus differentiation rate was 5.7%.