Genetic Evolution Of GST Gene Family In G. Barbadense And Its Key Orthologous Genes' Resistance To Verticillium Wilt

Verticillium wilt is one of the most serious diseases to affect the cotton yield and fiber quality.The onset of verticillium wilt is occurred about at the true leaf stage with 3-5 leaves,and it is outbreak from bud stage,then plants can wilt or die.The cause of verticillium wilt is V.dahliae and V.albo-atrun to infect cotton.And the most efficient ways to resistance verticillium wilt are cloning genes,parsing the molecular mechanism of verticillium wilt resistance,improving the genetic basis,and breeding resistant varieties in cotton.Glutathione S-transferase(GST)genes belong to a multigene family in organisms.The function of GST is cell detoxification by catalyzing glutathione to bind the intracellular toxic substances and to pump toxin out of cells.In previously study,we proved that GST is vital to resist verticillium wilt in G.hirsutum.But,the cognition of GST and the function of verticillium wilt resistance are unknown and need be further studied in G.barbadense to date.Here,the evolution mechanism of GST genes were analyzed in G.barbadense,including to identify the genes number,to locate the genes,to analyze the gene structure.The GbGST16930 on D-subgenome 09 chromosome was silenced to clear the verticillium wilt resistance in G.barbadense.As the orthologous gene of GbGST16930,the verticillium wilt resistance and molecular mechanism of GhGST1510 were researched in G.hirsutum.The main results were as follows:1.A total of 123 GST genes were found in G.barbadense,including tau,DHAR,Eflb?,Lambda,MAPEG,Phi,TCHQD,Theta,and Zeta classes.They were located on 25 chromosomes except D-subgenome 06 chromosome,and the greatest number was tau class.For 123 GST genes,61 were located on A-subgenome and 62 were located on D-subgenome.And 14 tandems were found in G.barbadense,including 13 tandems of tau.The intron number was one for almost all of tau class,and the number of introns were from two to 16 for other eight GST classes.Six ancient GST clusters were identified on A-subgenome 02,09,and 13 chromosomes and on A-subgenome 02,09,and 13 chromosomes by a circle mapping in G.barbadense,and most of which were tau class genes.Especially,one tau cluster(including GbarD09G016910,GbarD09G016920,and GbarD09G016930)was integrated on D-subgenom 09 chromosome in G.barbadense.It was not like the orthologous cluster on D-subgenom 09 chromosome in G.hirsutum,which only remained GhDt09G1520 due to gene dropout event.But,the orthologous cluster was complete on A-subgenom 09 chromosome in G.hirsutum.The result was important for revealing the different molecular basis between G.barbadense and G.hirsutum.2.For identifying verticillium wilt resistance function of GbGST16930(GbarD09G016930),the GbGST16930 was silenced by VIGS method in G.barbadense.The silence efficiency was 70%.The disease indexes(DI)were 46 and 23 respectively for VIGS plants and wild type plants under V.dahliae stress,which indicated that the expression of GbGST16930 could enhance the verticillium wilt resistance in G.barbadense.3.As the orthologous gene of GbGST16930,GhGST1510(MK179292)was cloned.And the ORF of GhGST1510 was 651 bp,encoding 216 AA.Two conserved domains were identified for GhGST1 510,including GST_N_Tau and GST_C_Tau domains.4.Under V.dahliae stress,the DIs were 32.8 and 82.8 for over-expression 35s:GhGST1510 plants and wild type plants in Arabidopsis thaliana,respectively.Which indicated that the expression of GhGST1510 could enhance the verticillium wilt resistance in Arabidopsis thaliana.The prokaryotic expression analysis showed that a 24.6 kda peptide was obtained.The subcellular localization analysis showed that GhGST1510 was located in the cell membrane,nucleus and cytoplasm.Eleven possible interacting proteins with GhGST1510 were identified by yeast two-hybrid.Among of them included CIPK3,ABCI19,GATA9 and MYB330.Which indicated that GhGST1510 maybe participated in ABA and SA synthesis pathways.In conclusion,a total of 123 GST genes were identified in G.barbadense.And the difference evolution of one cluster was found on D-subgenome 09 chromosome between G.hirsutum and G.barbadense.The expression of GbGST16930 could enhance verticillium wilt resistance by VIGS assay in G.barbadense.And as the orthologous gene of GbGST16930,the expression of GhGST1510 could enhance verticillium wilt resistance by over-expression assay in Arabidopsis thaliana.The GhGST1510 was located in the cell membrane,cytoplasm and nucleus.Eleven possible interacting proteins with GhGST1510 were identified by yeast two-hybrid.These proteins were participated in ABA and SA synthesis pathways.