| Verticillium wilt,which mainly induced by soil-borne fungus Verticillium dahliae,is one of the most serious threaten in cotton fiber industry.RING type E3 ubiquitin ligase RDUF was involved in plant defense response to diseases,growth and development,and abiotic stresses.In the present study,RING E3 ubiquitin ligase GhRDUF4D was cloned from Gossypium hirsutum,and its expression patterns and disease resistance response were evaluated.This study will provide theoretical basis for elucidating the involvement of RING E3 ubiquitin ligase in plant disease resistance.The results are as follows:1.A total of 6,7,14 and 14 RDUF(RING-DUF1117)genes were detected in Gossypium arboreum,G.raimondii,G.hirsutum and G.barbadense,respectively.Gene structure and domain analysis showed that RDUF genes in each group were highly conserved.Gene duplicated analysis indicated that the segmental duplication and whole genome duplication(WGD)events were occurred during cotton evolution.Many environment stress and hormone response related cis-elements were detected in GhRDUF promoters region.The predicted target transcription factors and miRNAs implied the extensive role of GhRDUFs in diversiform stresses.2.In order to explore the function of RDUF gene in upland cotton,the identification and expression characteristics of RDUF gene in cotton were analyzed,GhRDUF genes were response to cold,drought and salt stresses,and sensitize to JA,SA and ET signals.The expression of GhRDUF4 A,GhRDUF6 A and GhRDUF7 A were up-regulated significantly under drought stress.After SA spraying,the expression of GhRDUF2 A and GhRDUF4 A were significantly up-regulated.The expression of GhRDUF4D,GhRDUF5 A and GhRDUF7 A were significantly up-regulated under ET treatment.The results indicated that RDUF genes were involved in environment adaption and multiple hormones response.3.The expression of GhRDUF4D was up-regulated significantly upon V.dahliae infection.Overexpression of GhRDUF4D in Arabidopsis increased theresistance of V.dahliae in transgenic plants.Compared with control,GhRDUF4D silenced plants were more sensitive to V.dahliae in Upland cotton,showing severe symptoms of leaf wilt and abscission.Meanwhile,the amount of V.dahliae was significantly increased in stems of GhRDUF4D silenced cotton plants.The function of GhRDUF4D against Verticillium wilt was confirmed.4.In this study,GhRDUF4D gene was introduced into Upland cotton by agrobacterium-mediated genetic transformation.Through callus induction,embryogenic callus induction,tissue embryo induction and rooting induction,a total of 30 resistant cotton plants in 7 lines were obtained,which 28 positive transgenic plants were examed by PCR method.The plants provide materials for molecular mechanism analysis of GhRDUF4D in further study. |
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