半胱氨酸蛋白酶抑制剂(cystatins)在提高作物对生物和非生物胁迫的抗性等方面具有重要作用，但其分子机制及调控途径尚不清楚。项目组前期研究发现，苹果基因组中cystatins基因的表达受干旱等多种非生物胁迫诱导，富平楸子MpCYS2和MpCYS4在拟南芥中过量表达能提高其抗旱性。酵母双杂筛选到一个与MpCYS4互作的蛋白—MpFER1。在此基础上，本项目拟通过BiFC等手段进一步验证MpCYS4和MpFER1 的相互作用并筛选与MpCYS2互作的蛋白；通过对MpCYS2和MpCYS4原核表达、亚细胞定位、转化苹果，进一步鉴定其在苹果干旱应答中的功能；利用MpCYS4和MpFER1干扰和过量表达的苹果转基因材料，分析干旱下MpCYS4对ABA信号的调控并通过转录组测序分析其调控通路。其目的是探明cystatins在干旱胁迫中的功能及分子调控机制，为植物抗旱机理和抗逆改良提供新的理论依据。

Plant cystatins play an important role in enhancing tolerance of both biotic and abiotic stress, but the molecular mechanisms and regulatory pathway by which cystatins respond to stresses remains unclear. Our previous data demonstrated that the expression of apple cystatin genes is induced by drought and other abiotic stress. Meanwhile, ectopic expression of MpCYS2 and MpCYS4 from Malus prunifolia conferred drought tolerance in Arabidopsis. We also identified an interacting partner of MpCYS4 using Yeast Two-Hybrid screening, MpFER1. In this project, we will (1) verify the interaction between MpCYS4 and MpFER1 by BiFC and isolate the interacting partners of MpCYS2 in M. prunifolia by Yeast Two-Hybrid screening assay, (2) perform inhibition assay of cysteine protease activity by MpCYS2 and MpCYS4 fusion protein in vitro, examine their subcellular localization, and over-express or silence them in apple to investigate the functions of MpCYS2 and MpCYS4 under drought stress in apple. To investigate how MpCYS4 is involved in ABA signaling pathway and the regulatory network under drought stress, we will characterize the phenotypes and transcriptome of transgenic apples overexpressing or silencing MpCYS4 and MpFER1. The ultimate goal of this project is to reveal the function and regulatory mechanisms of cystatins (MpCYS2 and MpCYS4) during drought stress in Malus plants, and provide new theoretical basis for plant drought resistance mechanism and genetic breeding for stress resistance.