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Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (04): 593-600.

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Identification of Promoter Region of GhWRKY64Induced by Multi-stresses in Cotton (Gossypium hirsutum)

DU Hao,DING Lin-Yun,HE Man-Lin,CAI Cai-Ping,GUO Wang-Zhen*   

  1. State Key Laboratory of Crop Genetics & Germplasm Enhancement, Hybrid Cotton Research & Development Engineering Research Center, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China
  • Received:2014-10-17 Revised:2015-02-06 Online:2015-04-12 Published:2015-03-02
  • Contact: 郭旺珍, E-mail: moelab@njau.edu.cn, Tel: 025-84396523

Abstract:

WRKY proteins are members of a transcription factor family with Zinc-finger structure in higher plant, which play diverse roles in plant responses to stresses and in various physiological processes.A 1064bp promoter sequence of GhWRKY64 was isolated from Gossypium hirsutum acc. TM-1, and its regulatory elements and functional characterization were further analyzed. Bioinformatics analysis showed that there were 18 putative tissue-speci?c or stress-induced regulatory motifs corresponding to known cis-elements in eukaryotic genes, including six ROOTMOTIFTAPOX1 of root-specific regulatory elements, two W-boxes, four CACTFTPPCA1mesophyll-specific regulatory elements, four OSE2ROOTNODULE of pathogenic bacteria-induced elements, and two GTIGMSCAM4 of salt regulatory elements. Further, pGhWRKY64 promoter region was fused to a β-glucuronidase(GUS) reporter gene as pBIW64:GUS constructed for investigation of important regions controlling gene expression, and 12 transgenic tobacco plants were obtained by Agrobacterium-mediated leaf-disc transformation method. Further, pBIW64-5 line with the highest GUS expression was selected for different tissues/organs expression and induced expression analyses. Histochemical staining in seedlings stage of the transgenic tobacco plants showed that the full-length promoter directed ef?cient expression of the reporter gene in the root and leaf, however, no GUS activity was detected in non-transgenic control.When transgenic tobacco plants growed at the flowering stage,GUS activity in root, leaf and petiole was detected, especially in root and root tip with heavier staining than those in other tissues, but no GUS activity was detected in stem and flower tissues. After treatment with Verticilliumdahliae, pBIW64:GUS showed greater induction and stronger GUS activity than those in untreated transgenic tobacco plants. Taken together, pGhWRKY64 promoter with 1064bp iselite regulatory element for preferential accumulation of foreign genes in root and leaf tissues and stress responses, which will be used for transgenic research in cottonVerticillium dahliae-resistant breeding.

Key words: GhWRKY64promoter, Abiotic and biotic stress, Transgenic tobacco, Regulatory elements, GUS activity

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