Physiological And Molecular Mechanisms Of Resistance To Powdery Mildew In Red Clover(Trifolium Pratense L.),Cloning And Genetic Transformation Of TpGDSL Gene

Red clover(Trifolium pratense L.)is one of perennial legumes with nutritional value and yield second only to Alfalfa(Medicago sativa L.),which is widely used and has broad development and application prospects.Powdery mildew(Erysiphales),as a kind of common and important obligate biotrophic pathogen,can seriously reduce the yield of red clover and limit its application and development in grass and animal husbandry.In order to elucidate the effects of powdery mildew on physiological and biochemical characteristics,endogenous hormones and cell structure of red clover,and to verify the function of tpgdsl gene for resistance to powdery mildew,the powdery mildew susceptible cultivar(Minshan red clover)and the powdery mildew resistant cultivar(Minshan red clover)were studied×The resistance of F2 hybrids of powdery mildew resistant varieties(Gannong RPM1 red clover)to powdery mildew was evaluated,and the powdery mildew resistant population was established.The physiological and biochemical indexes,endogenous hormone content and cell structure of individual plants in different resistant populations were determined by artificial inoculation;The resistant and susceptible plants of F₂ population were used as materials for transcriptome analysis;The candidate gene TpGDSL related to powdery mildew resistance of red clover was cloned by transcriptome analysis,and the over expression vector PHB-GDSL was constructed and transformed into Arabidopsis thaliana.The main results are as follows1.The pathogen is Erysiphe trifoliorum.After artificial inoculation,the electrical conductivity(EC)of resistant materials increased at first and then decreased,while that of susceptible materials increased continuously.The EC of susceptible materials increased by 3.57 times after15 days of inoculation.The relative water content(RWC)of resistant materials decreased first and then increased.When the susceptible materials continued to decrease,the RWC of susceptible materials decreased by 31.85%after 15 days of inoculation.The activities of superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and soluble sugar(WSC)increased 11 days and 7 days after inoculation,respectively,and then decreased,with the highest values of 534.43±10.07 U·g^-1·min^-1 FW,411.73±4.08 U·g^-1·min^-1 FW,136.53±1.00U·g^-1·min^-1 FW and 32.02±0.57 mg·g^-1 FW,respectively.The contents of malondialdehyde(MDA)and free proline(Pro)increased by 4.84 and 5.38 times respectively.The contents of zeatin(ZR),jasmonic acid(JA)and salicylic acid(SA)in resistant materials showed two peaks on the first and seventh days after inoculation,and the contents of zeatin(ZR),jasmonic acid(JA)and salicylic acid(SA)in susceptible materials increased from the first day before inoculation to the first day after inoculation,and then decreased continuously.The content of ABA in resistant materials decreased after artificial inoculation,but the change trend was opposite in susceptible materials.The contents of ZR,JA,SA and ABA were the highest on the7th,1st,7th and 1st day after inoculation,and their values were 12.23±1.27 ng·g^-1,15.55±0.30ng·g^-1,124.82±1.68 ng·g^-1,483.50±125.50 ng·g^-1,respectively,which were 3.52,0.93,1.60and 1.04 times higher than those before inoculation.After inoculation,the change range of various hormones in the leaves of resistant red clover was larger than that of susceptible red clover,which indicated that the effect of endogenous hormones induced by powdery mildew was more obvious in resistant red clover.In this study,it was found that the endogenous hormones in red clover leaves were related to their resistance to powdery mildew,but the correlation and cross cooperation process and mode of these hormones in resistance to powdery mildew need to be further explored.2.The width of upper epidermis cells,thickness of leaves,thickness of palisade tissue and wax content of resistant red clover leaves were significantly higher than those of susceptible red clover leaves(P<0.01),and the differences between resistant and susceptible red clover leaves were 16.13%,22.29%,29.99%and 85.90%respectively.After powdery mildew infection,the width of upper epidermis cells increased,palisade tissue thickened and palisade tissue cells arranged more closely and orderly,while the thickness of spongy tissue increased significantly and spongy cells arranged loosely and disorderly in susceptible materials.Resistant red clover can resist the invasion of pathogenic hyphae by increasing the thickness of leaf and palisade tissue and compressing the cell space of palisade tissue.The content of hemicellulose,cellulose and lignin in leaves was increased by powdery mildew infection,and the content of soluble pectin was decreased.The content of cellulose,hemicellulose,lignin and hydroxyproline glycoprotein in the resistant materials after infection was higher than that of susceptible materials and control materials.Powdery mildew infection induced physical defense of red clover,and each component played a positive role in the process of resistance to powdery mildew.3.Difference of resistance after infection of powdery mildew The DEGs were enriched in phenylpropane pathway,formaldehyde-valerate pathway,lignin and lignans pathway,and glucosinolates pathway,respectively.CHRs,C2H2,had,MYB,b ZIP and MADS are involved in the defense response of red clover powdery mildew.AXR1,CYP,cand1 and PPR like genes in SA and IAA pathway may play a positive role in the defense response of red clover powdery mildew.There were 23,20,18 and 14 DEGs in cytochrome P450,oxidase,phosphatase,nitrilase and GDSL lipase family.PAL,C4H,4CL and BGL in lignin metabolism pathway,NAD(P)-binding Rossmann fold in ABA regulation pathway and 12 oxo PDA in gibberellin metabolism pathway are involved in the defense process of red clover.Transcriptome analysis showed that the gdsl homologous gene of Trifolium pratense had high background expression and differential expression multiple.4.A red clover TpGDSL gene encoding 366 amino acids and 1101 bp in length was cloned.The similarity between the GDSL gene of Medicago truncatula and the gene was 86.47%.The molecular formula of TpGDSL gene is C₁₇₇₆H₂₇₀₄N₄₇₀O₅₆₁S₁₅,the relative molecular weight is40.9672 KD,the theoretical isoelectric point(PI)is 4.39,the positive and negative charge residues are 20 and 35,the instability coefficient is 31.38,and the fat coefficient is 81.01.TpGDSL protein may exist in the extracellular matrix.The protein mainly consists of 36.34%alpha helix,4.10%beta turn,17.49%extended strand and 42.08%random coil,which were covered by two-stage structure and three-dimensional structure,and the coverage and reliability were 82%and 100%respectively.In this study,the recombinant overexpression vector PHB GDSL was transferred into wild type Arabidopsis thaliana by Agrobacterium mediated inflorescence staining method,and 16 lines T₁ generation positive transgenic seeds were obtained,which laid a foundation for the functional analysis of TpGDSL gene.