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Functional Analysis of Cotton Anti-Verticillium Wilt Gene GhERF and Transformation Research of Anti-Verticillium Wilt Related Gene GhHRGP

Author: WengYu
Tutor: DaiXiaoFeng
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Cotton Resistance-related Genes Genetic transformation Resistance Functional Analysis
CLC: S435.621
Type: Master's thesis
Year: 2008
Downloads: 128
Quote: 1
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Abstract

Verticillium wilt is difficult to control of soil-borne vascular disease, seriously affecting cotton production through conventional cross-breeding methods are difficult to obtain both have excellent resistance to verticillium wilt and agronomic traits of the species. Isolated using molecular biology techniques to study the cotton verticillium wilt resistant gene, and through transgenic technology to obtain cotton verticillium wilt resistant features and traits, is a way of having prospects. In this study, two related genes resistant to Verticillium wilt GhERF (Gossypium hirsutum Ethylene Responsive Factor gene) and GhHRGP (Gossypium hirsutum Proline-Rich Glycoprotein gene) full-length cDNA sequences, the use of this laboratory dahliae toxin-induced cotton seedling clones income. Amino acid sequence alignment showed that, GhERF specific ethylene plants belonging to family of transcription factors, and Arabidopsis ethylene response element binding protein AtEBP (Ethylene-Responsive Element BindingProtein) homology of 45%, similar in structure to Arabidopsis ERF mustard family subfamily B-2. GhHRGP protein may be involved in the composition of plant cell walls, its composition and hydroxyproline-rich glycoprotein (Hydroxyproline-Rich Glycoprotein) similar. Ethephon Verticillium wilt resistance in Upland Cotton Variety 12 of 7-day-old seedlings, using semi-quantitative RT-PCR studies GhERF and GhHRGP expression induced by ethylene. The results show that, GhERF in root tissues showed nearly constitutive expression characteristics, and its expression levels did not change significantly compared with the control, while in the leaf tissue of the basal expression level is low, 2h to 12h after ethylene treatment significantly increased expression levels, Description GhERF in the roots and leaves have different tissue expression, and its expression levels in leaf tissue greatly influenced by ethylene. GhHRGP root and leaves in the control group in each of the periods into the expression was not detected by ethephon expression not detected after treatment, indicating that the root and leaves GhHRGP tissues induced from ethylene. Constructed GhERF and GhHRGP two genes of the plant expression vector, respectively, using Agrobacterium-mediated cotton (CCRI 24, CCRI 713 394 and Hebei together) and Arabidopsis (Col-0) were GhERF and GhHRGP genetic transformation. GhHRGP Transformation of Cotton (CCRI 24) won eight germ; GhERF transformed Col-0 won GhERF overexpressing transgenic Arabidopsis plants homozygous. To verify the resistance in Arabidopsis genes in cotton resistance identification, using a variety of methods studied VD991 V. dahliae toxins and spore suspension of Arabidopsis Col-0, Nd-1, C24 and WS four eco-pathogenic situation, the establishment of research and Arabidopsis dahliae interaction experiment platform. The results show that identification of toxins seedling and adult vaccination needle suitable for transgenic Arabidopsis disease resistance against bacterial wilt research. In a sterile seedling identification of toxins found in 10μg/ml ~ 20μg/ml of toxins within the four kinds of Arabidopsis ecotypes showed different degrees of leaf wilting, chlorosis symptoms, including Nd-1 and C24 most sensitive to the toxin, Col-0 moderately sensitive, WS is relatively insensitive to the toxin. Inoculated with spore suspension acupuncture adult leaves, there will be varying degrees of wilting, chlorosis and wilt symptoms such as yellowing. In GhERF homozygous transgenic Arabidopsis plants as materials, toxins were identified using transgenic plants for large dahliae resistance and GhERF regulatory functions were analyzed. At a concentration of 12.5μg/ml under the conditions GhERF overexpressing transgenic Arabidopsis plants homozygous for resistance identification of toxins that transgenic plants and wild-type leaves are wilting symptoms appear, the statistical results of diseased plants were 50 percent and 71 percent, respectively, the disease index was 12.5% ??and 30.9%. Show GhERF overexpression transgenic plants increased tolerance to the toxin. Reported in the literature by GhERF regulation 8 downstream defense genes for semi-quantitative RT-PCR analysis showed that, in addition to the expression level of PR1 and Thi2.1 no change compared with the control outside, PR2, PR3, PR4, PDF1.2, PRXR1 and Di19 6 genes expression levels of performance increase, which PDF1.2, PR3 and PR4 expression levels increased significantly, indicating GhERF with ethylene signaling pathway downstream regulated gene expression in defense capabilities, while the salicylic acid signaling pathway and jasmine acid signaling pathways downstream defense genes PR1 and Thi2.1 expression may not be affected. This indicates GhERF is an ethylene signal transduction pathways involved in and has a regulatory function of important plant defense response candidate genes resistant to Verticillium wilt.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Economic crop pests and diseases > Fiber crop pests and diseases > Cotton pests and diseases > Disease
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