棉花GhGT-2转录因子的克隆及其功能分析

doi:10.3864/j.issn.0578-1752.2015.24.002

Abstract

Abstract: 【Objective】Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stresses during its growth season and its productivity is significantly limited by adverse factors. Trihelix transcription factors are important proteins involved in response to abiotic stresses in plants. Understanding the molecular mechanisms of the Trihelix transcription factor genein cotton will lay the foundation for improving the stress tolerance of cotton by gene manipulation.【Method】We searched the cotton ESTs database using BLAST (the Basic Local Alignment Search Tool). One GT unigene, named GhGT-2, was obtained. Sequence analysis of GhGT-2 was performed to confirm that this gene is a member of the Trihelix GT-2 subfamily gene. The total RNA from the leaves of cotton was used as the template to design the degenerate primers based on expressed sequence tags. The full-length cDNA of GhGT-2 were cloned using the method of Rapid amplification of cDNA Ends (RACE) combined with reverse transcription-PCR (RT-PCR). Homologous analysis and multiple alignments were performed with MEGA 5. SMART online tools were used for protein sequence analysis. Seedlings were treated with conditions simulating drought, salinity, cold and abscisic acid (ABA) at the 15-day-old seedling stage in Xin luzao 26. After treatment for 0 , 1 , 3 , 6 , and 12 h, the seedlings were harvested respectively. In the meantime, cotton roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA) were harvested at different growth periods. The expression profiles of the GhGT-2 in various tissues and under cold, drought, salt and ABA treatments were investigated by quantitative real-time PCR (qRT-PCR) analysis. The GhGT-2【Result】The full-length cDNA sequence was obtained successfully from upland cotton, which was 1 579 bp in lenght, containing a 1 428 bp open reading frame that encoded a protein of 476 amino acids with a predicted molecular weight of 54.07 kD and an isoelectric point of 8.96. SMART analysis showed GhGT-2 contained two typical SANT motifs of GT-2 subfamily. Phylogenetic analysis showed that GhGT-2 belonged to the GT-2 subfamily of the Trihelix family and was most closely related to AtGTL1 and PtaGTL1. Real-time quantitative PCR (qRT-PCR) analysis revealed that GhGT-2 constitutively expresses in all tested tissues, such as roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA). The expression level of GhGT-2 was highestin leaves, and was very low in roots. The expression profiling revealed that GhGT-2 was induced by ABA, high salt and drought at 3, 6, and 12 h points, but was weakly repressed at 1 h point. In contrast, expression of GhGT-2 was weakly repressed at 3, 6, and 12 h points under cold treatment apart from the 3 h point. These results suggested that GhGT-2 might be involved in stress resistance-related response of ABA signaling pathways in cotton plant. Using the Arabidopsis protoplasts assay system, we found that GhGT-2 protein was located in cell nuclei but its trans-activation activity could not be detected clearly. Electrophoretic Mobility Shift Assay (EMSA) showed that GhGT-2 protein could bind to GT2-Box, GT3-Box, GT-1b (BoxⅡ), MBS1, MRE1, MRE3 and MRE4. 【Conclusion】A full-length cDNA of GhGT-2 was isolated and the typical SNAT binding domain were found in the deduced protein, belonging to the Trihelix GT-2 subfamily. The expression of GhGT-2 was up-regulated under drought, high salt and ABA treatments, showing that GhGT-2 was involved in the ABA dependent stress-responding gene network. It was deduced that GhGT-2 may play important roles in upland cotton plant abiotic stress adaption. coding sequence was cloned onto GFP expressing vector pBI221, and GAL4 DNA-binding domain vector, transformed into Arabidopsis protoplasts to verify the localization in the cell and the transcriptional activation ability of the GhGT-2 protein. The gel-shift assay was performed to detect DNA binding elements.

Key words: cotton (Gossypium hirsutum （EMSA）L.), Trihelix transcription factor, abiotic stress, subcellular localization, trans- activation activity, electrophoretic mobility shift assay

LI Yue, LIU Xiao-dong, XIE Zong-ming, DONG Yong-mei, WU Dong-mei, CHEN Shou-yi. Cloning and Function Analysis of Cotton Transcription Factor GhGT-2[J].Scientia Agricultura Sinica, 2015, 48(24): 4872-4884.

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Cloning and Function Analysis of Cotton Transcription Factor GhGT-2

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