番茄小热激蛋白CI-sHSP17.7对果实糖代谢低温抑制的分子调控机制

Small heat shock proteins (sHSPs) play an important role in the process of adverse environmental stresses, and it is also important for regulation of fruit growth and metabolism. However, it is still unclear about the specific protection targets in fruit development and how to cause the accumulation of sugar and enhance low temperature resistance in the fruits. We have characterized a small heat shock protein gene, CI-sHSP17.7, from transgenic tomato plants constitutive expressing invertase inhibitor (INH) by RNA-Seq, when plants were grown under low temperature, transgenic fruits accumulated sugar earlier than controls, and conferred enhanced resistance to low temperature in ripening fruits. In the present study, CI-sHSP17.7 will be analyzed in the subcellular localization, tissue-specific expression patterns and response under low temperature stress by Real-time quantitative PCR and CI-sHSP17.7-GFP DNA fusion. CI-sHSP17.7 will be also used as baits in yeast two-hybrid (YTH) assays to screen a tomato fruit cDNA library and found to interact with the target. Interactions will be confirmed by pull-down and BiFC assays. CI-sHSP17.7 gene will also be constitutively or inhibited expressed in the different kinds of sucrose accumulation tomatoes via Agrobacterium-mediated transformation. The regulation role of sugar metabolism of CI-sHSP17.7 in fruit development and low temperature stress will be studied in ripening fruits. Thus, these lines can be proposed as promising candidates for breeding programs aimed to improve cultivated tomato fruit yield and quality under low temperature stress.

作为应对不良环境胁迫产生的特定抗逆蛋白sHSPs，对调控果实生长发育及代谢也具有重要意义，但其在果实发育中的具体保护靶标及其在低温下如何引起果实糖分积累、增强耐低温性的机制尚不清楚。本项目在已明确了果实高表达的Class I sHSP 17.7KD与低温下糖含量变化的基础上，一方面通过实时定量PCR及启动子的GUS-GFP分析获得低温下CI-sHSP17.7基因的时空表达及亚细胞定位特征；并以CI-sHSP17.7作为诱饵筛选番茄果实cDNA表达文库，获得候选的靶标蛋白，为分析sHSPs途径在果实逆境响应中的功能打下基础；另一方面对不同蔗糖积累型番茄进行抑制与过表达的转基因分析，探讨CI-sHSP17.7在番茄果实成熟过程中调控蔗糖代谢的分子机理及与低温抑制的关系，为番茄耐低温育种及品质的遗传改良提供新思路。

番茄作为喜温性茄果类蔬菜，遭遇低温胁迫会造成品质受损、产量下降等伤害，带给种植者不可估量的经济损失。sHSPs不仅在热胁迫等逆境下具有保护细胞的分子伴侣属性，而且一些sHSPs在果实发育及冷藏期间以及冷藏后的恢复过程起重要作用，但 sHSPs 在植物生长发育中的确切作用及耐冷的分子机制目前还不清楚。作为应对不良环境胁迫产生的特定抗逆蛋白sHSPs，对调控果实生长发育及代谢也具有重要意义，但其在果实发育中的具体保护靶标及其在低温下如何引起果实糖分积累增强耐低温性的机制尚不清楚。前期工作中课题组明确了番茄果实中高表达的ClassI-sHSP17.7与低温下糖含量变化，本项目首先对小热激蛋白家族的生物信息学分析及家族成员的表达特性分析，通过实时定量PCR及启动子的GUS-GFP分析获得低温下CI-sHSP17.7基因的时空表达及亚细胞定位特征；并以CI-sHSP17.7作为诱饵筛选番茄果实cDNA表达文库，获得候选的靶标蛋白，通过体内双分子荧光互补（bimolecular fluorescence complementation, BiFC）和体外GST-pull down分析，验证其与SlHSP17.7存在互作，这为分析sHSPs途径在果实逆境响应中的功能打下基础。另一方面本研究分别在己糖积累型的栽培番茄与蔗糖积累型的野生番茄中进行了基因的过表达与抑制表达的遗传转化，并获得了稳定的转基因材料。通过低温处理分析，明确了栽培番茄中高水平的SlHSP17.7和低水平的SlCCX-LIKE样蛋白可减轻低温胁迫下程序性细胞死亡(PCD)，SlHSP17.7可能是SlCCX1-like靶向内质网(ER)膜蛋白的辅助因子，通过维持细胞内Ca2+稳态，降低冷应激敏感性。研究成果为番茄耐低温育种及品质的遗传改良提供了新思路，也为进一步阐明果菜类蔬菜的抗逆性研究奠定了理论基础。