Functional Analysis Of Cotton Anti-Verticillium Wilt Gene GhERF And Transformation Research Of Anti-Verticillium Wilt Related Gene GhHRGP

Verticillium wilt is a soilborne vascular disease which is difficult to control and harmful to cotton production severely.It is difficult to get the anti-Verticllium and good agricultural characters together by normal breeding.Utilizing the technology of molecular biology,the Verticillium resistance-related gene can be isolated easily,therefore it is a prospective way to breed the anti-Verticilllium lines with new functional characters by the technology of gene transfer.The full cDNA sequences of the two Verticillium resistance-related genes GhERF and GhHRGP, were obtained by 5′RACE through the inducement of cotton seedlings by VD toxin.The results of BLASTp indicated that GhERF belongs to a plant specific gene family of Ethylene-Resoponsive factor, which shares 45%homology with the AtEBP(Ethylene-Resoponsive Element Bingding Protein) and shows the structrual similarity with the B-2 subfamiliy of the ERF family in Arabidopsis.GhHRGP seemed to have the function involving in the protein of the plant cell wall constitution,whose constitution is similar with the hydroxyproline-rich glycoprotein.Treating the 7d seedlings of upland cotton cultivar Zhongmian 12 with ethephon,the expression pattern of GhERF and GhHRGP induced by ethylene was studied employing the semi-quantitative RT-PCR.The results indicated that GhERF was expressed nearly consitutively in root,its expression level did not change except for the slightly up-regulating 2 and 12 hpi.The background expression level of GhERF was low in leaves,and was up-regulated steadily from 2 to 12 hpi,which infered the different expression pattern of GhERF in roots and leaves and showed more ethylene-influced pattern in leaves. The expression of GhHRGP could be detected in roots and leaves neither in control nor in treatment of ethephon,which indicated that the expression of GhHRGP in roots and leaves was not induced by ethylene.Using the two plant expression vector constructs of GhERF and GhHRGP,the Agribacterium-mediated transformation was carded out on cotton(Zhongmian 24,Zhongmian 394 and Jihe 713) and Arabidopsis(Col-0).8 plantules were obtained through the transformation of Zhongmian 24 by GhHRGP and the overexpression transgenic homozygotes were obtained through the transformation of Col-0 by GhERF.In order to identify the resistance of the resistance-related gene in Arabidopsis,several methods were employed to research the resistance of Arabidopsis ecotype Col-0,Nd-1,C24 and WS to VD toxin and VD spore suspension,the pathosystem platform between Verticillium and Arabidopsis was primarily established.The results indicated that the methods of toxin-seedling interaction and puncture inoculation on mature leaves were highly evaluated to be employed in the resistance identification of the Arabidopsis transgenic plants.By the experiments of toxin-seedling interaction on medium,it was observed that in the range of 10μg/ml～20μg/ml all four ecotypes showed the symptoms of wilt and chrolosis to different extents,among which Nd-1 and C24 were the most susceptible to the toxin,Col-0 was the moderate one and WS was comparably not susceptible to the toxin.The wilt and chrolosis symptoms of mature leaves treated by puncture inoculation were in different degrees.Using the Arabidopsis GhERF transgenic homozygotes as materials,the GhERF function of regulation and resistance to Verticillium dahliae was identified.The resistance identification of the GhERF overexpression transgenic homozygotes was carried out by using toxin-seedling system.The transgenic homozygotes showed wilt symptom similar with the control,but diseased plant rate and disease index of the transgenic homozygotes were 50%and 12.5%respectively lower than 71%and 30.9%of the control.These results indicated the tolerance of the transgenic homozygotes to the toxin was enhanced in comparison with control at the level of 12.5μg/ml.Expression level of 8 downstream defense genes reported to be regulated by GhERF were detected by employing the semi-quantitative RT-PCR,the results showed that the expression level of PR2,PR3,PR4,PDF1.2,PRXR1 and Di19 were up-regulated,especially for PDF1.2,PR3 and PR4,except for the expression level of PR1 and Thi2.1,which were not changed in comparison with the control.So it was indicated that GhERF was capable of regulating the expression of defense genes downstream of the ethylene signaling pathway, and maybe not involved in the regulation of defense genes PR1 and Thi2.1 downstream of the SA signaling pathway and JA signaling pathway.It could be concluded from the results that GhERF was an important candidate resistance gene which was involved in ethylene signalling pathway and capable of regulating the plant defense response.