Cloning And Preliminary Function Verification Of TreFQR1 In White Clover

White clover（Trifolium repens L.）is a good perennial leguminous grass widely cultivated in worldwide,and drought,high temperature and other stresses will cause its peroxidative damage,limiting its normal growth and development.TrFQR1 belongs to the family of anthraquinone reductase superfamily that is dependent on FMN.It exists in various organisms and has detoxification effect on quails,which can respond to oxidative stress and reduce oxidative damage.In this study,RACE and RT-PCR methods were used to clone and obtain the full-length quinone oxidoreductase family protein gene TrFQR1 which was FMN-dependent and NADPH-dependent in the white clover of’Latino’,and the bioinformatics analysis of TrFQR1 was performed.The q RT-PCR technique was used to analyze the gene under abiotic stress（high temperature,low temperature,drought,salt,and heavy metal stress）,hormones（ABA,CTK,IAA,and Spm）and signal molecules(NO,H₂S,H₂O₂,and Ca²⁺).Expression pattern;At the same time,construct the expression vector of TrFQR1 gene,transform Agrobacterium,transform Arabidopsis thaliana into Agrobacterium tumefaciens inoculation method,test the positive plants by PCR,and use screening medium to obtain transgenic Arabidopsis homozygous T3 plants.The main results are as follows:1)In this study,the full-length c DNA of the TrFQR1 was 1 003 bp,and the open reading frame（ORF）was 612 bp,encoding 203 amino acids.The protein has a molecular formula of C₉₉₃H₁₅₂₉N₂₅₁O₂₉₀S₈,a molecular weight（MW）of 21.88 k Da,and an isoelectric point theory p I of 5.96.TrFQR1 protein is a stable hydrophilic protein,no signal peptide,no transmembrane structure,no disordered features.The phosphorylation site predicted that there were 7 serine（Ser）phosphorylation sites,2 Tyr phosphorylation sites,and 8 threonine（Th）phosphorylation sites in TrFQR1.TrFQR1 has a Flavodoxin-like domain.In the secondary structure of TrFQR1,α-helix accounts for 49.26%of the total,random coil accounts for 26.6%,and extended strand andβ-turn account for 14.29%respectively.And9.85%.Homology comparison and phylogenetic tree analysis showed that the white truffle Tre FQR1protein sequence shares high similarity with other leguminous plants.Subcellular localization results showed that the TrFQR1-encoded protein is mainly localized in small organelles.2)Under high temperature stress,the relative expression of TrFQR1 gene was up-regulated after treatment for a period of time in leaves,reached a maximum at 3 h,and then decreased in roots after a period of treatment,reaching a minimum at 1.5 h;under low-temperature stress,in leaves,The relative expression level of TrFQR1 gene was down-regulated after a period of treatment,reached a minimum at6 h,and was up-regulated at roots after a period of treatment and reached a maximum at 3 h.Under salt stress and heavy metal stress,the relative expression of TrFQR1 gene in leaves and roots changed significantly.The relative expression of TrFQR1 was up-regulated in leaves,increased first in roots,then decreased,and reached the maximum at 6h and 1.5h,respectively.Under PEG stress,the relative expression of TrFQR1 gene in leaves and roots changed significantly and the trend was consistent,reaching the maximum at 3h.Among 5 abiotic stresses,the relative expression of TrFQR1 gene had the most significant change under high temperature stress.3)In SNP,H₂O₂ and Ca Cl₂ treatment,the relative expression of TrFQR1 gene was more significant in the root,and it was up-regulated after treatment for a period of time,reached the maximum at 1.5h,6h and 6h respectively;the expression level of the root was also increased.,reached maximum at 1.5h,6h and 1.5h,respectively.In Na HS treatment,the relative expression level of TrFQR1 gene was more significant in the roots,its expression was up-regulated and reached its maximum at 6 h.In leaves,the expression level of TrFQR1 was down-regulated and reached a minimum at 1.5 h.Under Spm,ABA,and CTK treatment,the relative expression of TrFQR1 was more significant in leaves than in leaves.After treatment for a period of time,the expression of TrFQR1 was up-regulated and reached the maximum at 1.5h,3h,and 1.5h,respectively.The relative expression of TrFQR1 was detected under IAA treatment.The changes were more pronounced in the roots,and the expression was up-regulated,reaching a maximum at 6 h.Among them,the relative expression level of TrFQR1 gene changed most significantly under IAA treatment.4)Using Columbia type 0（Col-0,wild-type）Arabidopsis thaliana as a test material.After 37 days of normal growth,compared with wild-type Arabidopsis,the overexpression of OE3 and OE7 plants showed no significant difference in the morphological size of aerial parts,but the number of fibrous roots increased significantly;over-expressed plants OE3,OE7 and wild-type Arabidopsis thaliana were exposed to salt stress for 9 days.The leaf wilting did not show significant difference;on 16 d drought stress,the relative water content of over-expressing plant OE3 and OE7 was significantly higher than that of wild-type Arabidopsis thaliana,and the drought resistance was better.The relative OE3 and OE7plants contained over-expressed 10 d after high temperature stress.The amount of water was significantly higher than that of wild-type Arabidopsis thaliana.The relative electrical conductivity and malondialdehyde content were significantly lower than those of the wild type and were more resistant to high temperatures.