Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (6): 1097-1105.doi: 10.3864/j.issn.0578-1752.2016.06.006

• PLANT PROTECTION • Previous Articles     Next Articles

Screening and Identification of the Interacting Protein of Cytokinin Response Regulator VvRR2 in Grapevine

YU Yi-he, LI Xiu-zhen, GUO Da-long, YANG Ying-jun, LI Xue-qiang, ZHANG Guo-hai   

  1. College of Forestry, Henan University of Science & Technology, Luoyang 471003, Henan
  • Received:2015-11-24 Online:2016-03-16 Published:2016-03-16

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Abstract: 【Objective】The objective of this study is to clone cytokinin response regulator VvRR2 in grapevine, obtain the interaction protein of VvRR2, and elucidate the mechanism of VvRR2 in the disease resistance response in Vitis vinifera. 【Method】 Powdery mildew was inoculated on grapevine, total RNA extraction and reverse transcription, the transcripts of VvRR2 response to powdery mildew was detected by real-time quantitative PCR at transcription level. The transient expression vector pBI221-VvRR2- GFP was constructed, Arabidopsis protoplasts was transformed and the subcellular localization was analyzed. The yeast expression vector pGBKT7-VvRR2 was constructed and transformed into yeast strain AH109, and the transcriptional activity of VvRR2 was detected. The yeast cDNA library was constructed, VvRR2 was used as a bait to screen the interaction protein by Mating method. The candidate sequence was analyzed by Blast. The candidate protein VvTGA sequence was cloned into pGADT7 vector to form recombinant vector pGADT7-VvTGA, with the recombinant bait plasmid pGBKT7-VvRR2 were transformed into yeast, VvRR2 and VvTGA were verified with yeast two-hybrid methods. VvTGA full-length sequence was cloned into pSPYNE (R) 173 vector to form the recombinant vector pSPYNE-VvTGA, VvRR2 full-length sequence was cloned into pSPYCE (M) vector to form the recombinant vector pSPYCE-VvRR2. Then, the two recombinant vectors were co-transformed into Arabidopsis protoplasts, VvRR2 interaction with VvTGA were verified with bimolecular fluorescence complementary technology.【Result】After inoculation of the powdery mildew, the expression pattern of VvRR2 was regulated by the powdery mildew. The VvRR2 was located in the nucleus of Arabidopsis protoplasts, and the transcriptional activation experiment results showed VvRR2 had transcriptional activation activity in yeast. On the culture medium containing 60 mmol?L-1 3-AT, the self-activating activity of VvRR2 bait vector could be inhibited, and the bait vector of VvRR2 had no toxicity to the host yeast. VvRR2 was used as the bait to screen the yeast cDNA library, and 287 clones were obtained. The Blast analysis showed that these genes were involved in protein synthesis and degradation, signal transduction, light reaction and biological clock rhythm, growth development and the stress tolerance. The yeast two-hybrid assays showed that the yeast containing empty vector (pGADT7 or pGBKT7) in four deficient medium (containing 3-AT) couldn’t grow. However, the yeast containing two kinds of recombinant plasmid in four deficient medium (containing 3-AT) could grow, also showed color in four deficient medium (containing X-α-Gal). Bimolecular fluorescence complementation assays results showed co-transformation pSPYCE-VvRR2 and pSPYNE (R)173, pSPYNE-VvTGA and pSPYCE (M) into protoplasm, the transformed protoplasm had not yellow fluorescence. However, the transformed protoplasm containing pSPYCE-VvRR2 and pSPYNE-VvTGA recombinant vectors showed yellow fluorescent. The expression of VvTGA was similar to VvRR2, presented by pathogen induced expression patterns. 【Conclusion】 Grapevine cytokinin response regulator VvRR2 is a transcription factor, the expression is induced by powdery mildew, VvRR2 interacts with VvTGA, and the expression of VvTGA induced by powdery mildew.

Key words: grapevine (Vitis spp.), cytokinin response regulator, VvRR2, VvTGA, interaction protein

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