葡萄细胞分裂素响应调节因子VvRR2互作蛋白筛选与鉴定

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

【目的】从葡萄中克隆细胞分裂素响应调节因子VvRR2,获得VvRR2的互作蛋白,为阐明VvRR2在欧洲葡萄抗病反应中的作用机制提供依据。【方法】对葡萄接种白粉病菌,提取总RNA后反转录,利用实时荧光定量PCR检测VvRR2转录本对白粉病菌的响应;构建瞬时表达载体p BI221-VvRR2-GFP,转化拟南芥原生质体进行亚细胞定位分析;构建酵母表达载体p GBKT7-VvRR2,转化酵母菌株AH109,检测VvRR2的转录激活活性;构建酵母表达c DNA文库,以VvRR2为诱饵,通过Mating法筛选互作蛋白,对获得候选序列进行Blast分析;将候选蛋白VvTGA的全长序列克隆至p GADT7载体形成重组载体p GADT7-VvTGA,与重组诱饵载体p GBKT7-VvRR2共转化酵母,进行双杂交验证VvRR2与VvTGA的相互作用;将VvTGA的全长序列克隆至p SPYNE(R)173载体,形成重组载体p SPYNE-VvTGA,将VvRR2的全长序列克隆至p SPYCE(M)载体,形成重组载体p SPYCE-VvRR2,然后将两个重组载体共转化拟南芥原生质体,利用双分子荧光互补技术验证VvRR2与VvTGA的相互作用。【结果】葡萄接种白粉病菌后,细胞分裂素响应调节因子VvRR2呈现受白粉病菌诱导表达模式。VvRR2定位在拟南芥原生质体的细胞核,转录激活试验结果表明VvRR2在酵母体内具有转录激活活性。在含有60 mmol·L-1的3-AT培养基上可以抑制VvRR2诱饵载体的自激活活性,VvRR2诱饵载体对宿主酵母菌没有毒性。以VvRR2为诱饵,初步筛选到287个单克隆,在高严谨条件下进一步筛选获得23个有效序列,Blast分析显示这些基因参与蛋白质合成与降解、细胞分裂素信号传导、光反应和生物钟节律、生长发育和逆境响应。酵母回复双杂交试验结果显示含有空载体(p GADT7或p GBKT7)酵母在四缺培养基(含3-AT)上不能生长,含有两种重组质粒的酵母在四缺培养基(含3-AT)上能够生长,并在含有X-α-Gal的四缺培养基上能够显色。双分子荧光互补试验结果显示共转化p SPYCE-VvRR2与p SPYNE(R)173、p SPYNE-VvTGA与p SPYCE(M)的原生质体没有黄色荧光,而共转化p SPYCE-VvRR2与p SPYNE-VvTGA的原生质体显示黄色荧光。VvTGA的表达类似于VvRR2,呈现受白粉病菌诱导表达模式。【结论】葡萄细胞分裂素响应调节因子VvRR2是一个受白粉病菌诱导表达的转录因子,能够与VvTGA相互作用,并且VvTGA受白粉病菌诱导表达。

【Objective】The objective of this study is to clone cytokinin response regulator VvR R2 in grapevine, obtain the interaction protein of Vv RR2, and elucidate the mechanism of Vv RR2 in the disease resistance response in Vitis vinifera. 【Method】 Powdery mildew was inoculated on grapevine, total RNA extraction and reverse transcription, the transcripts of Vv RR2 response to powdery mildew was detected by real-time quantitative PCR at transcription level. The transient expression vector p BI221-Vv RR2-GFP was constructed, Arabidopsis protoplasts was transformed and the subcellular localization was analyzed. The yeast expressionvector p GBKT7-Vv RR2 was constructed and transformed into yeast strain AH109, and the transcriptional activity of Vv RR2 was detected. The yeast c DNA library was constructed, Vv RR2 was used as a bait to screen the interaction protein by Mating method. The candidate sequence was analyzed by Blast. The candidate protein Vv TGA sequence was cloned into p GADT7 vector to form recombinant vector p GADT7-Vv TGA, with the recombinant bait plasmid p GBKT7-Vv RR2 were transformed into yeast, VvR R2 and Vv TGA were verified with yeast two-hybrid methods. Vv TGA full-length sequence was cloned into p SPYNE（R） 173 vector to form the recombinant vector p SPYNE-Vv TGA, Vv RR2 full-length sequence was cloned into pS PYCE（M） vector to form the recombinant vector p SPYCE-Vv RR2. Then, the two recombinant vectors were co-transformed into Arabidopsis protoplasts, Vv RR2 interaction with Vv TGA were verified with bimolecular fluorescence complementary technology.【Result】After inoculation of the powdery mildew, the expression pattern of Vv RR2 was regulated by the powdery mildew. The VvR R2 was located in the nucleus of Arabidopsis protoplasts, and the transcriptional activation experiment results showed Vv RR2 had transcriptional activation activity in yeast. On the culture medium containing 60 mmol·L-1 3-AT, the self-activating activity of VvR R2 bait vector could be inhibited, and the bait vector of Vv RR2 had no toxicity to the host yeast. Vv RR2 was used as the bait to screen the yeast c DNA 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（pG ADT7 or p GBKT7） 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 p SPYCE-Vv RR2 and p SPYNE（R）173, p SPYNE-Vv TGA and pS PYCE（M） into protoplasm, the transformed protoplasm had not yellow fluorescence. However, the transformed protoplasm containing p SPYCE-Vv RR2 and p SPYNE-Vv TGA recombinant vectors showed yellow fluorescent. The expression of Vv TGA was similar to VvR R2, presented by pathogen induced expression patterns. 【Conclusion】 Grapevine cytokinin response regulator Vv RR2 is a transcription factor, the expression is induced by powdery mildew, Vv RR2 interacts with Vv TGA, and the expression of Vv TGA induced by powdery mildew.