Function Of GbVWR1 From Gossypium Barbadense In Verticillium Wilt Resistance

Verticillium wilt is one of the major diseases of cotton,which poses a great threat to cotton yield and fiber quality.Gossypium hirsutum and G.barbadense are the two largest cultivated varieties in the world.Among them,G.hirsutum has high yield and strong adaptability,but it lacks thevarieties with immunity or high resistance to Verticillium wilt.The G.barbadense has the characteristics of immunity or high resistance to Verticillium wilt and good quality.Therefore,screening resistant genes and analyzing resistant mechanism are significant for cotton molecular breeding.In the former research,our research group constructed the full-length cDNA library of?G.barbadense?Pima90-53induced by V.dahliae and obtained an unknown gene named GbVWR1?VWR=Verticillium wilt resistance?.It was preliminarily confirmed that the gene was involved in the resistance to Verticillium wilt.In order to further study the function of GbVWR1 resistance to Verticillium wilt,GbVWR1 was transformed into Arabidopsis thaliana for overexpression using transgenic technology.The resistance mechanism of the gene was preliminarily explored from physiology,biochemistry and molecular biology.Furthermore,the interaction proteins with GbVWR1 were screened from two-hybrid cDNA library of island cotton induced by V.dahliae based on yeast two-hybrid system,and the resistance mechanism of interaction proteins was discussed.The main results were as follows:1 The overexpression vector of GbVWR1 was successfully constructed and transformed into Arabidopsis thaliana.The homozygous lines of transgenic A.thaliana were obtained.The results of semi-quantitative RT-PCR and Western Blot showed that the GbVWR1 protein was successfully expressed in the transgenic lines.The disease index showed that L1 reduced by 24%,L2 reduced by 25%,L3 reduced by 27%when compared with that of WT.Further analysis of significant differences revealed GbVWR1was significantly lower than that of WT control.Therefore,overexpression analysis showed that GbVWR1 increased the plant resistance to V.dahliae.2.Under V.dahliae stress,at 12 hpi and 24 hpi,the H₂O₂ content of transgenic A.thaliana was significantly higher than that of WT.POD activity was significantly higher than that of WT at 12 hpi,but POD activity between transgenic Arabidopsis and WT were not significant at 24 hpi.It was possible to increase plant resistance by affecting the H₂O₂ content and POD activity and participated in their signal pathways.3.RT-PCR showed that the marker gene NDR1 and EDS1 of SA pathway were significantly higher than that of WT,and up-regulated expression after induced by V.dahliae.The expression of NPR1,PR1,PR5 was significantly lower than that of WT,and the expression of three genes in transgenic Arabidopsis thaliana was down-regulated expression after induced by V.dahliae.This may indicate that GbVWR1 may induce a large accumulation of SA in the regulation of SA signaling pathway,but excessive SA accumulation may inhibit the SA-NPR1-PR.The expression of marker gene in JA,ET and BR signaling pathway in transgenic Arabidopsis thaliana was significantly higher than that of WT.The marker gene PR4 of JA pathway and the marker gene ERF1 of ET signaling pathway down-regulated expression;The maker gene PDF1.2 of JA/ET signaling pathway and the marker gene BAK1 of BR signaling pathway up-regulated expression.It is suggested that GbVWR1 is involved in multiple signal pathways.4.The bait vector of GbVWR1 was constructed and transformed into yeast receptive cell Y2Hgold.It is proved non-toxic and self-activated.The vector was hybridized with yeast two-hybrid cDNA library.Twenty-four proteins interacting with GbVWR1 were screened out from two-hybrided cDNA library.They included ABCG36 of ABC transporter family,ACO3 gene which regulated ethylene signal transduction pathway and involved in ethylene synthesis,and GH3 gene,which is involved in salicylic acid-mediated plant defense response.5.The yeast hybrid showed that the plasmids with pGBKT7-GbVWR1 and pGADT7-ABCG36 could not only grow normally but also the colony could turn blue in QDO/X/A media,which indicated that there was interaction reaction between ABCG36and GbVWR1.In conclusion,GbVWR1 was involved in cotton resistance to V.dahliae and may be an important positive regulatory factor.It may be involved in multiple signaling pathways by interacting with multiple proteins.