小麦锌指转录因子TaDi19A对低温的响应及其互作蛋白的筛选

Response of Wheat Zinc-Finger Transcription Factor Ta Di19A to Cold and Its Screening of Interacting Proteins

【目的】锌指类转录因子在植物逆境信号转导和非生物胁迫响应中发挥重要的作用。通过对小麦锌指转录因子基因Ta Di19A的耐冷性能进行鉴定,利用酵母双杂交技术筛选并获得与Ta Di19A互作的候选蛋白,以解析Ta Di19A介导的抗逆调控机制。【方法】通过对低温处理的小麦转录组测序结果进行分析,获得一个锌指类转录因子Ta Di19A。利用生物信息学的方法分析Ta Di19A的分子特性,用SMART在线工具进行蛋白结构分析;用GSDS和PHYRE2在线工具分别对Ta Di19A结构和蛋白三级结构进行分析;用Net Phos 2.0 Server数据库预测Ta Di19A蛋白磷酸化位点。以低温处理的小麦c DNA作为模板,通过SYBR Green染料法进行实时荧光定量PCR,检测Ta Di19A在低温处理不同时间段的表达模式。构建植物表达载体p BI121-Ta Di19A,通过花序侵染法转化拟南芥,用T3代拟南芥进行耐冷性鉴定,分析低温处理对转基因拟南芥的根长、鲜重和存活率的影响。检测转Ta Di19A拟南芥中抗逆相关基因表达变化,分析Ta Di19A调控植物耐冷性的作用机制。构建诱饵载体p GBKT7-Ta Di19A,验证自激活活性;利用酵母双杂交技术,将诱饵载体p GBKT7-Ta Di19A和小麦c DNA文库共转化酵母AH109感受态细胞,通过SD/-Trp/-Leu/-His/-Ade和X-α-gal显蓝反应筛选得到阳性克隆,测序和BLAST分析获得候选蛋白。【结果】小麦Ta Di19A编码区全长747 bp,编码248个氨基酸,分子量为28.03 k D,等电点为4.74,基因含4个外显子,3个内含子。Ta Di19A蛋白靠近N-端包含锌指结合结构域,C端为Di19结构域,预测的Ta Di19A蛋白三级结构包含2个α螺旋结构。磷酸化位点分析结果显示Ta Di19A蛋白含有12个丝氨酸、9个苏氨酸和3个酪氨酸磷酸化位点。实时荧光定量PCR结果显示,Ta Di19A受低温胁迫诱导表达。正常生长条件下,转基因和野生型拟南芥没有明显差异,低温处理下,转基因拟南芥的根长明显大于野生型拟南芥,并且耐冷性强于野生型拟南芥。下游基因检测结果表明,低温处理后,CBL1、CBL2和KIN1等冷胁迫响应相关基因在野生型和转基因植株中表达量都升高,在转基因植株中的表达量显著高于野生型,表明Ta Di19A可能通过调节下游冷胁迫响应相关基因的表达提高转基因植物的耐冷性。通过对酵母双杂交系统筛选到的候选互作蛋白进行初步分析表明,这些候选互作蛋白主要参与植物体的信号转导和非生物胁迫响应过程,表明Ta Di19A在植物的逆境信号转导及非生物胁迫响应过程中发挥着重要作用。【结论】小麦Ta Di19A受低温诱导表达,过表达能够提高转基因拟南芥的耐冷性;而Ta Di19A功能的发挥可能需要其他蛋白的参与。

【Objective】Zinc-finger transcription factors play an important role in stress signal transduction and abiotic stress response in plants. In this study, the function of Ta Di19 A was identified under low temperature stress and its interacting proteins were screened by yeast two-hybrid system to explore the regulation mechanism of Ta Di19 A.【Method】Ta Di19 A gene was isolated from the cold-treated wheat transcriptome profile. Bioinformatics method was used to analyze the molecular properties of the Ta Di19 A gene, SMART online tools were used for protein structure analysis; GSDS and PHYRE2 online tools were used to analyze gene structure and tertiary structure of Ta Di19 A protein; Net Phos 2.0 Server database was used to predict phosphorylation sites of Ta Di19 A protein. The quantitative real-time PCR（q RT-PCR）, conducted using the cold-treated wheat c DNA based on SYBR Green technology, was used to analyze the expression pattern of Ta Di19 A under cold temperature stress treatment in different time periods. Ta Di19 A was fused with PBI121 to transform into wild-type（WT） Arabidopsis plants（Columbia-0） mediated by the floral dip method, homozygous T3 seeds of transgenic lines and WT were used for cold tolerance analysis which the root length, fresh weight, and survival rate were measured before and after cold treatment. The expressions of four stress-response genes were investigated in transgenic lines and WT under normal and low temperature conditions to analyze the cold-resistant regulation mechanism of Ta Di19 A. Bait plasmid p GBKT7-Ta Di19 A was constructed and the self-activation was detected. The p GBKT7-Ta Di19 A and wheat c DNA library was co-transformed into yeast cell AH109 by two-hybrid system, and the positive clones were screened via SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/X-α-gal plate and these single clones were sequenced and analyzed by BLAST to obtain the interaction candidate proteins.【Result】The full length of Ta Di19 A gene was 747 bp with 4 exons, encoding 248 amino acids, and the protein molecular weight and isoelectric point of were 28.03 k Da and 4.74, respectively. Ta Di19 A protein included Zinc-finger binding domain, Di19 domain and the predicted tertiary structure contained 2 alpha helix. Phosphorylation site analysis showed that there were 12 serine, 9 threonine, and 3 tyrosine phosphorylation sites in Ta Di19 A protein. q RT-PCR analysis showed that Ta Di19 A was induced by low temperature. The root length and cold tolerance assays revealed that Ta Di19 A transgenic Arabidopsis increased the cold tolerance. The expression of several cold-stress-responsive genes was monitored through PCR analysis, the expression of genes CBL1, CBL2, CBL3 and KIN1 showed elevated levels in both WT and transgenic Arabidopsis plants under cold-stress condition, and the expression levels in transgenic plants were significantly higher than those in WT. Analysis of candidate proteins screened by yeast two-hybrid system revealed that those proteins mainly affected the signal transduction and abiotic stress response, which demonstrated that Ta Di19 A is critical to stress signal transduction and abiotic stress response in plants. 【Conclusion】Cold-inducible Ta Di19 A improved cold tolerance in transgenic Arabidopsis; Ta Di19 A might work via interacting with other proteins.