Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (9): 117-125.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0090

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Screening of OsCRK5-interacted Proteins in Rice Using Yeast Two-hybrid System

WANG Zi-ying(), LONG Chen-jie, FAN Zhao-yu, ZHANG Lei()   

  1. College of Life Sciences, Wuhan University, State Key Laboratory of Hybrid Rice, Wuhan 430072
  • Received:2023-02-07 Online:2023-09-26 Published:2023-10-24
  • Contact: ZHANG Lei E-mail:747262414@qq.com;arabilab@whu.edu.cn

Abstract:

Rice(Oryza sativa L.)is an important food crop, research on the regulation mechanism of rice growth and development can lay a theoretical foundation for the improvement of rice varieties. Calcium dependent protein kinases(CDPKs)are important protein kinases in plants which participate in plant growth and development, as well as in response to environmental reactions. CRK5(CDPK related kinase 5)in rice is highly homologous to CDPK in protein sequence and structure. Yeast two-hybrid screening library technology was used to screen OsCRK5 interacting proteins related to plant drought resistance in order to explore the molecular mechanism of OsCRK5 participating in drought response in rice plants. Firstly, the 1-1 332 bp fragment of OsCRK5 was cloned into the plastmid pGBKT7, and the bait vector pGBKT7-OsCRK5 was obtained. After verification by sequencing, the bait vector was transformed into the yeast strain Y2H Gold.It was observed that the recombinant protein didn't demonstrate the toxic effects and self-activation in selective nutrition medium, and the expression of the recombinant protein was analyzed by Western blot. Furthermore, the interacting proteins of OsCRK5 were screened from the rice cDNA library and 77 positive clones were obtained. The functional prediction showed that the interacting proteins were involved in protein synthesis, degradation and storage process, transcriptional regulation, cell growth and division process, energy metabolism and cell metabolic processes. Finally, OsWR1 and OsDi19-1 related to plant drought stress response were selected from positive clones. The interaction between OsCRK5 and OsWR1 and OsDi19-1 was verified by yeast two-hybrid and biomolecular fluorescence complementation experiments. The results lay a foundation for the genetic improvement of drought tolerance in rice.

Key words: rice, OsCRK5, yeast two-hybrid system, cDNA library, drought stress