G蛋白γ亚基CsGG3.2调控黄瓜低温响应的机制研究

Cucumbers, an important economic crop widely cultivated, are sensitive to low temperature. Their tissues, fruits and even whole plants suffer physiological obstacles under low temperature conditions, especially when they are cultivated in greenhouses during the winter and early spring. Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in eukaryotes. Recently, G protein has been shown to be involved in the activation of cold-induced Ca2+ signaling in plant. However, it’s not will establish that how G protein triggers Ca2+ signaling. Plant Gγ subunits, divided into A, B, and C three structurally distinct types, provide functional specificity to the heterotrimer complex. We found that the expression of CsGG3.2, a C type Gγ gene in cucumber, is induced by low temperature, and we hypothesized that CsGG3.2 may play an important role in triggering Ca2+ signaling under low temperature. Transgenic and physiological approaches will be used to characterize the functionality of CsGG3.2 in low temperature response and triggering Ca2+ signaling for chilling tolerance in cucumber. This study will have great theoretical and applicable significance in revealing molecular basis of cold response of cucumber and mitigating low temperature-induced cucumber loss under greenhouse conditions.

黄瓜是我国设施栽培的主要蔬菜，不耐低温，冬春季节易受冷害。G蛋白是一类重要的信号传导分子，参与植物低温信号的响应过程，与钙信号的产生有关。但低温下G蛋白激活植物钙信号的机制尚不明确。γ亚基对于植物G蛋白功能的发挥极其重要。本项目在初步探明黄瓜Gγ基因CsGG3.2对低温响应的基础上，研究CsGG3.2在G蛋白激活响应低温的钙信号过程中的作用。拟构建CsGG3.2的过表达和RNAi载体，并转化黄瓜，研究钙信号阻断物质和钙信号增强物质对转基因植株低温耐受性的影响，明确转基因与野生型黄瓜在低温响应过程中细胞内游离Ca2+的积累、分布、Ca2+流特征的差异，揭示CsGG3.2调控黄瓜低温响应的机制，为通过调控G蛋白信号增强黄瓜耐冷性提供理论依据。

G蛋白参与植物低温响应过程，与钙信号产生有关，但机制尚不明确。γ亚基对于植物G蛋白功能的发挥极其重要，本项目研究了黄瓜C型Gγ在G蛋白激活响应低温的钙信号中的作用。构建了C型Gγ基因过表达、反义和RNAi载体，获得了CsGG3.2和CsGG3.1-2的过表达和抑制表达植株。过表达CsGG3.2增强了黄瓜低温耐受力，上调了CsCBF1、CsCBF2及CsCOR15b和CsKIN1的表达，表明CsGG3.2可通过CBF途径正调控低温响应；抑制表达CsGG3.1-2则降低了黄瓜的低温耐受性，但CBF及靶基因的表达未受抑制，表明CsGG3.1-2通过非CBF途径调控低温响应，并且CsGG3.1-2和CsGG3.2两种Gγ在调控低温响应中不仅有协同作用，而且具有差异性。转录组分析发现CsGG3.1-2抑制表达植株的钙信号转导受到影响：2个与类钙调磷酸酶B亚基CBL互作的蛋白激酶CIPK基因（其中CIPK7的同源蛋白调控拟南芥的低温响应）、1个类钙调蛋白CML基因和另外2个钙结合蛋白基因的表达受到抑制，表明Gγ可能通过CIPK7调控钙信号途径参与低温应答。研究中发现抑制CsGG3.1-2表达导致黄瓜叶绿素含量降低、叶片黄化。转录组分析发现1个叶绿素降解基因下调，12个叶绿素合成基因上调，另有定位于叶绿体的23个核糖体基因、5个氨酰tRNA合成酶基因、5个FTSH蛋白酶基因、1个Clp蛋白酶基因和2个参与叶绿体蛋白运输的基因上调；此外2个液泡Fe转运蛋白基因、1个K转运蛋白基因和1个参与Fe-S簇合成的SufE2同源基因下调。利用Micro-XRF技术和常规方法对元素的测试结果显示转基因黄瓜叶片K和Fe含量降低。表明抑制CsGG3.1-2表达可能通过影响K和Fe吸收进而造成铁硫簇合成减少，致使光合相关蛋白及叶绿素合成受阻。研究结果为揭示G蛋白调控植物低温响应和生长发育的机制提供了依据。