Cloning And Characterization Analysis Of ERF Sub-family Transcription Factor Gene From Cotton (Gossypium Hirsutum L.)

Object: Sea island cotton, also known as long staple cotton, which fiber is slender, shiny and strong andalso is an important material for spinning top-grade and special textile. Xinjiang is the only production areaof Sea Island cotton in China. But in recent years the disease caused by Fusarium oxysporum f. sp.Vasinfectum（Fov）has not been well controlled in Sea Island cotton and the disease has been expanding andincreasing yearly. Due to Sea Island cotton Fov resistance gene resource was not rich and the traditionalmethod of distant hybridization breeding has the shortage of big separation and stable slow, which all madethe process of Fov resistance varieties’ breeding slowly. ERF sub-family is a specific class of transcriptionfactors in plants. They can activate or inhibit expression of downstream defense genes to change diseaseresistance of plants through the interaction of ERF and GCC-box cis-acting elements. So the object of thisresearch was the screening and cloning of ERF-B3subgroup transcription factor gene related to Fovresistance from upland cotton, which can offer new gene resource and theoretical basis for Sea Islandcotton Fov resistance varieties’ improvement by genetic engineering.Methods: In this research “Zhongmiansuo12” was selected as the test material, which was a high resistantcultivar to the infection of Fov. In the course of in silico cloning, BLAST searches of cotton EST databasewere performed using the AP2/ERF domain of the ERF-B3subgroup transcription factors fromArabidopsis thaliana as a query sequence; The expression model of cloned genes were analyzed at differenttime after the treatment of Fov by semi-quantitative RT-PCR. Then, GhB301, an ERF-B3subgrouptranscription factor gene related to Fov resistance was screened and cloned by RT-PCR. The expressionmodel of this gene was analyzed by qPCR after the treatments of Fov, ET, SA, drought, high salt and lowtemperature. To further explore the function of this gene, a plant expression vector was constructed andtransformed.Results:1. Based on the NCBI database information and related softwares, through the method of in silico cloningand RT-PCR,15B3subgroup gene specific fragments were amplified. Semi-quantitative RT-PCR analysisshowed that four genes such as GhB301, GhB302, GhB305and GhB307can response to the inducement ofFov and the responsion of GhB301was the strongest.2. By the technology of RT-PCR, GhB301(KC222015), a new ERF-B3subgroup transcription factor generelated to Fov resistance was cloned from the roots of “Zhongmiansuo12” which was a high resistantcultivar to the infection of Fov. Sequence analysis indicated that the full length cDNA of GhB301was593bp, with384bp ORF encoding127amino acids and a conserved AP2/ERF domain;3. qPCR analysis indicated that, after the treatment of Fov, the expression level of GhB301in resistancecultivar was obvious higher than those of susceptible cultivar and the time to the expression peak inresistance cultivar was also earlier than susceptible cultivar. The expression level of GhB301in roots was higher than stems and leaves under the treatment of Fov. ET and SA can induce the expression of GhB301and the responsion to ET lasted longer than SA. GhB301gene can also response to drought, lowtemperature and high salinity. But the responsion to low temperature was stronger than the others.4. Bioinformatics analysis showed that protein encoded by GhB301gene was hydrophilic, free oftransmembrane regions and signal peptides; the main components of secondary structure was α-helix andirregular curls; typical AP2/ERF domain located in18-76amino acids and with the highest possibilitylocated in the nucleus.5. GhB301gene over-expression vector was constructed. Using the method of Agrobacterium mediation weconducted the genetic transformation of tobacco.13over-expression transgenic tobacco plants wereacquired after the screening of PCR. Compared with the wild-type tobacco, the phenotype of transgenictobacco plants had not changed significantly, but the expression level of PR1and PR2elevated markedly.Conclusion: GhB301was a new Fov resistance related ERF-B3subgroup transcription factor gene. Itcould participate in the response of plants to the pathogenic-bacteria, hormone and abiotic stresses and alsocould express in tobacco and active parts of PRs’ expression. But the function of GhB301still needs furtherexploration.