Cloning And Functional Characterization Of GbLOX And GbAOS In Gossypium Barbadense Defense In Response To Verticillium Dahliae

Verticillium wilt caused by Verticillium dahliae is a soilborne vascular disease, which is difficult to control and harmful to the production of cotton severely. Researcher had reported that LOX and AOS were involved in plant defense response, so, to study the resistance mechanism of Verticillium wilt is important for resistance breeding in cotton. From a full-length c DNA library of roots from Gossypium barbadense Pima 90-53 inoculated with Verticillium dahliae, we isolated four genes, named Gb9-LOX, Gb13-LOX, Gb AOS and Gb AOS2. The research includes gene cloning and bioinformatics analysis, cloning and sequence analysis of promoters of these genes, detection of gene expression after Verticillium dahliae infection and effects of different exogenous hormones on gene expression, differential expression analysis and resistance functional analysis after gene silencing. The results were as follows:1.The ORFs of two lipoxygenases(Gb9-LOX, Gb13-LOX) and allene oxide synthase(Gb AOS, Gb AOS2) from the root of cotton were cloned by homologous sequence cloning and RT-PCR, which included 2598 bp, 2616 bp, 1491 bp, 1572 bp open reading frams(ORF).2.Bioinformatics analysis showed that the Gb9-LOX, Gb13-LOX are members of Lipoxygenase superfamily and contain two conserved active sites of PLAT_LH2 and lipoxygenase; Gb AOS and Gb AOS2 belong to P450 superfamily and contain a conserved active site of PLN02648; neither of them contained signal peptide or transmembrane domain, and were all hydrophilic protein.3.The promoter of Gb9-LOX and Gb AOS1 were cloned from Gossypium barbadense Pima 90-53, whose lengh were 1067 bp and 1212 bp. Bioinformatics analysis showed that the promoter of Gb9-LOX include cis-acting regulatory elements involved in the Me JA-responsiveness, defense and stress responsiveness and salicylic acid responsiveness, ethylene-responsive element, gibberellin-responsive element, and MYB binding site involved in drought-inducibility; the promoter of Gb AOS1 contained cis-acting regulatory element involved in auxin responsiveness, defense and stress responsiveness and salicylic acid responsiveness, ethylene-responsive element, and MYB binding site involved in flavonoid biosynthetic genes regulation.4.QRT-PCR analysis indicated that Gb9-LOX, Gb13-LOX, Gb AOS and Gb AOS2 m RNA levels were drastically up-regulated in roots of G. barbadense seedlings inoculated with V. dahlia or spraying with methyl-jasmonic acid. When spraying with salicylic acid or ethylene, Gb9-LOX and Gb AOS2 m RNA levels were drastically down-regulated in leaf.5.QRT-PCR analysis showed that Gb9-LOX, Gb13-LOX, Gb AOS and Gb AOS2 transcripts were expressed in all tissues of cotton, but the transcripts of Gb9-LOX, Gb13-LOX and Gb AOS1 were most abundantly in the root and much less in stem, and the expression level of Gb AOS2 was higher in stem than other tissues in Gossypium barbadense Pima 90-53.6.Silencing of Gb9-LOX, Gb AOS1 by VIGS enhanced cotton plants susceptibility to V. dahliae.