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作物学报 ›› 2014, Vol. 40 ›› Issue (10): 1756-1766.

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

拟南芥H+-焦磷酸化酶AVP1互作小GTP结合蛋白AtRAB的特性鉴定与功能分析

刘荣榜1,2,陈明2,*,郭萌萌2,司青林2,高世庆3,徐兆师2,李连城2,马有志2,尹钧1,*   

  1. 1 河南农业大学 / 小麦玉米作物学国家重点实验室 / 河南粮食作物协同创新中心, 河南郑州450002; 2中国农业科学院作物科学研究所 / 基因资源与基因改良国家重大科学工程 / 农业部麦类生物学与遗传育种重点实验室, 北京100081; 3北京杂交小麦工程技术研究中心, 北京100097
  • 收稿日期:2014-03-10 修回日期:2014-06-16 出版日期:2014-10-12 网络出版日期:2014-07-23
  • 通讯作者: 尹钧, E-mail: xmzxyj@126.com, Tel: 0371-63558203; 陈明, E-mail: chenming02@caas.cn, Tel: 010-82108789
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2013ZX08002-002)和国家自然科学基金项目(31201200)资助。

Characterization and Functional Analysis of a Small GTP-binding Protein AtRAB Interacting with H+-Pyrophosphatase AVP1 in Arabidopsis thaliana

LIU Rong-Bang1,2,CHEN Ming2,*,GUO Meng-Meng2,SI Qing-Lin2,GAO Shi-Qing3,XU Zhao-Shi2,LI Lian-Cheng2,MA You-Zhi2,YIN Jun1,*   

  1. 1 Henan Agricultural University / National Key Laboratory of Wheat and Maize Crop Science / Collaborative Innovation Center of Henan Grain Crops, Zhengzhou 450002, China; 2 Institute of Crop Science, Chinese Academy of Agricultural Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081, China; 3 Beijing Hybrid Wheat Engineering and Technology Research Center, Beijing 100097, China
  • Received:2014-03-10 Revised:2014-06-16 Published:2014-10-12 Published online:2014-07-23
  • Contact: 尹钧, E-mail: xmzxyj@126.com, Tel: 0371-63558203; 陈明, E-mail: chenming02@caas.cn, Tel: 010-82108789

摘要:

用拟南芥AVP1为诱饵采用膜蛋白酵母双杂交系统筛选拟南芥cDNA文库, 获得一个与AVP1互作的小GTP结合蛋白AtRAB。酵母互作试验表明, AVP1AtRAB存在互作; 双分子荧光互补试验(BiFC)证明, AVP1AtRAB能在质膜和细胞核上发生相互作用。野生型拟南芥(WT)AtRABAVP1的拟南芥突变体rabavp1在高盐胁迫条件下, 随着NaCl浓度的加大, 突变体rabavp1的表型变化相似, 相对于WT都表现为根长缩短, 侧根数减少; 在低磷处理条件下, 随着磷浓度的逐渐降低, 2个突变体的表型相似, 相对于WT同样表现为根长缩短, 总根面积减少, 侧根数减少; 在相同磷浓度条件下, 突变体rab对胁迫的反应比avp1; 在低钾处理条件下, 随着钾浓度的降低, 2个突变体的表型与在低磷胁迫处理条件下的表型相似。结果说明, AVP1AtRAB可以在细胞膜和细胞核上相互作用, AtRAB能够影响植物对离子的吸收, AVP1AtRAB的突变体在高盐、低磷和低钾等胁迫条件下表型变化相似, 证明了2个基因都正向调控植物对高盐、低磷和低钾胁迫的反应, 它们属于同一信号途径。

关键词: 氢离子焦磷酸化酶, 小GTP结合蛋白AtRAB, 酵母双杂交法, 蛋白互作, 抗逆反应

Abstract:

H+-Pyrophosphatase (H+-PPase) is an important proton transporter in plants. It cooperates with H+-ATPase and transport protons in vacuole or extracellular area to maintain a constant H+ gradient, which enables the transport of ions and other components (e.g. amino acids, carbohydrates). In the current research, AVP1 was applied to membrane proteins-based yeast two-hybrid system, and a small GTP-binding protein AtRAB was identified by screening the Arabidopsis cDNA library. The interaction between AVP1 and AtRAB was confirmed by interaction analysis in yeast. Bimolecular fluorescent complementation (BiFC) analysis suggested that AtRAB and AVP1 interaction took place in the plasma membrane and the nucleus. Phenotypes of wild type (WT) and Arabidopsis mutants of avp1 (AVP1) and rab (AtRAB) were compared under high-salt, low-phosphorus and low-potassium conditions. Under the high salt stress, avp1 and rab presented similar phenotypes along with the increase of NaCl concentration, which were characterized by shortened root length and reduced lateral root number in comparison with the WT. The decreasing phosphorus concentration also led to the phenotypes of shortened root length, reduced lateral root number and reduced total root area in the two mutants. Particularly, stronger response was observed in rab than in avp1 under the same phosphorus concentration. The potassium treatments resulted in similar phenotypes to those in phosphorus treatment. The results indicated that AVP1 can interact with AtRAB on the plasma membrane and the nucleus and further influence the absorption of ions in plants. The two mutants of avp1 and rab showed similar phenotypes, suggesting that both AVP1 and AtRAB positively regulate plant response to high salt, low phosphorus and low potassium stresses in the same signaling pathway.

Key words: H+-Pyrophosphatase, Small GTP-binding protein AtRAB, Yeast two-hybrid system, Protein interaction, stresses response

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