成熟期番茄果实cDNA文库的构建及SlHSP17.7互作蛋白的筛选

The Construction of cDNA Library during Maturation of Tomato Fruits and Screening of S1HSP17.7 Interaction Protein

植物小分子热激蛋白(small heat shock proteins, s HSPs)是一种多样、古老的蛋白家族,分子量大小普遍在12~42 k D之间,包含典型的C端、N端和α晶状体蛋白域。s HSPs作为一种重要的分子伴侣在植物生长发育和响应逆境胁迫中起着重要的作用。本研究通过构建成熟期番茄果实cDNA文库,筛选与SlHSP17.7互作的蛋白,进而探索SlHSP17.7在果实发育进程中的分子机制。以Micro-Tom番茄绿熟期、转色期和完熟期果实cDNA为模板,构建番茄果实c DNA文库。构建pGBKT7-SlHSP17.7诱饵载体筛选与其互作的蛋白,利用酵母双杂交实验验证互作关系。经检测番茄果实cDNA文库库容是2.2×10~6CFU,工作液细胞密度是3.6×10~7cell/mL,均一化程度>106,结果符合质控要求。将pGBKT7-SlHSP17.7诱饵载体转化Y2HGold酵母菌株检测发现,其无毒性和自激活现象,经筛选c DNA文库、测序及序列比对分析得到钙阳离子交换体SlCCX1-like蛋白,利用酵母双杂交实验验证发现二者存在互作关系。经PCR鉴定后,所建文库质量良好,文库的库容和文库滴度均符合建库要求,用pGBKT7-SlHSP17.7诱饵载体筛选得到互作蛋白SlCCX1-like,为进一步研究在果实发育进程SlHSP17.7的生物学功能提供了保障。

Small heat shock proteins(sHSPs) is a diverse and ancient family of proteins with molecular weights generally between 12 kD and 42 kD, including typical C-terminal, N-terminal and α-crystallin domain. As an important molecular chaperone, s HSPs plays an important role in growth and development and response to stress in plant. In this study, we constructed a c DNA library of mature tomato fruits and screened the protein which interacted with SlHSP17.7 to explore the molecular mechanism of Sl HSP17.7 in fruit development.The cDNA library of tomato fruit was constructed by using the c DNA of mature-green, breaker and red-ripening fruits of Micro-Tom tomato as template. The pGBKT7-SlHSP17.7 bait vector was used to screen interaction protein, and the yeast two-hybrid assay was used to verify the interaction. The storage capacity of tomato fruit cDNA library was greater than 2.2 ×10~6 CFU, the cell density of working fluid was 3.6 ×10~7 cell/m L, and the degree of normalization was greater than 106, the results were in accordance with the quality control requirements. The bait vector of pGBKT7-SlHSP17.7 was non-toxic and self-activating by transforming into Y2 HGold yeast strains. Furthermore, the calcium cation exchanger SlCCX1-like protein was obtained by screening cDNA library, sequencing and sequence alignment analysis. The yeast two-hybrid assay was used to verify the interaction relationship between the SlHSP17.7 and SlCCX1-like. The library quality was good, the library capacity and library titer were consistent with the requirements of database construction. SlCCX1-like was obtained by screened cDNA library using pG BKT7-SlHSP17.7 bait vector. To further study the biological function of the fruit development process Sl HSP17.7.