| The cell wall of plant is not only an important barrier to resist pathogen invasion, butalso an important place where the host and pathogen interaction. The host plant cellprevents pathogen to re-infect and spread through a series of resistance responses occurredin the cell wall, intercellular layer and cytoplasm, such as cell wall lignification, calloseaccumulation, the stretch of protein synthesis, and other pathways. In this study, threedifferent verticillium wilt resistance cotton varieties Pima90-53, Jimian20and Han208were tested by V. dahliae infection and VIGS mothed, and then the cell structure, thecontent of simple sugar、acid soluble lignin and acid insoluble lignin content，theexpression level of GhLAC were detected and analyzed in different cotton plants. GhLACwas cloned from the disease-resistant cotton varieties Jimian20and constructed into GFPfusion expression vector, overexpression vector and RNAi vector, respectively. Thedistribution of GhLAC protein was studied in transgentic Arabidopsis. We also studied thecontents of simple sugar、acid soluble lignin、acid insoluble lignin and lignin component inthe cell wall of transgentic Arabidopsis. The main results were as follows:1. In three cotton varieties with different resistance, Pima90-53, Jimian20and Han208, the content of AIL was negatively correlated with the disease index of the threecultivars (r=0.99991*).2. The ORF (1701bp) of GhLAC (gi: EU642559.1) was cloned from the cDNA ofJimian20. The genomic DNA (2401bp) of GhLAC including6introns was obtained fromgenomic DNA of Jimian20. Seven homologous gene of laccase were found and all locatedin chromosome9of G. raimondii genome.3. GhLAC protein belonged to the small group of named blueenzymes, and lacatedin cell wall. The transcription level of GhLaccase in Jimian20was significantly higher thanthat in Han208at any point of test time. The expression level reached the highest at8hrafter infection and maintained the high level within three days.4. Three vectors including GhLAC overexpression vector pGNLac, fusion expressionvector pCamLacGFP and RNAi vector ipCamLac were constructed, and then6,12and8transgenic Arabidopsis lines obtained, respectively.5. The silencing of GhLAC by VIGS mothed enhanced plant susceptibility to V.dahliae, and plants exhibited more severe wilting phenotype than the vector control plants. The disease index of GhLAC-VIGSed cotton was66.99, which was highly significantdifference to the value41.97from the vector-VIGSed cottons as control.6. The over-expression of GhLAC in Arabidopsis enhanced the resistance, andsilencing of GhLAC declined the Verticillium wilt tolerance. After being infected by V.dahliae, the vascular tissues were bloked more seriously than that in wild type or RNAilines; the contents of Klason lignin, total lignin and guaiacyl monomers (G monomers) intransgenic Arabidopsis over-expression lines were highly significant higher (P<0.01) thanthose of wild type inoculated with V. dahliae or not. Our results showed that GhLAC fromG. hirsutum not only was involved in lignin content alteration and Verticillium wiltresistance, but also more maintain the content of G-lignin and S-lignin in cell wall wheninoculated with V. dahliae.7. The plant expression vectors pCamE (gi: JX841315) were constructed. PlasmidpCamE offers eight restriction endonuclease sites at the multiple cloning sites, fourupstream of the promoter and three downstream of the terminator. The results showed thatvector pCamE was an economy and wide versatility vector, by which the target gene canbe modified efficiently and inherit stablly in transgenic plant with different objective. |
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