番茄低温响应转录因子NAC4的功能鉴定及调控机理研究

NAC transcription factors play vital roles in regulating plant growth and developmental processes as well as in response to abiotic stresses. In our previous study, a novel NAC transcription factor gene, NAC4 was differentially expressed between Solanum habrochaites and S.lycopersicum under cold stress, and there were some obvious differences in the deduced amino acid sequences of ShNAC4 and SlNAC4. Preliminary functional analysis indicated that the ShNAC4 overexpression transgenic plants were dwarf, unable to stand erect, and hypersensitive to cold stress as compared with the non-transgenic plants. Co-expression analysis showed that the expression of NAC4 is highly correlated with pectin methylesterase and ACC synthase gene. We surmised that NAC4 may negatively regulate cold tolerance by regulating cell wall development and/or ethylene biosynthesis in tomato. To further elucidate the function and regulatory mechanism of NAC4 in growth and development, and cold stress response in tomato, and reveal whether the differences in sequence between ShNAC4 and SlNAC4 would lead to a change in molecular and biological functions, the expression patterns, subcellular localization, transcriptional activation, the functions and regulatory mechanisms of ShNAC4 and SlNAC4 in response to cold stress, cell wall development，and ethylene biosynthesis will be compared and analyzed. Based on these, the relationship between cell wall development, ethylene biosynthesis, and cold tolerance will be further explored in this study.

NAC转录因子在调控植物生长发育及非生物逆境应答中发挥着重要作用。前期，我们发现低温胁迫下一新的NAC转录因子基因（NAC4）在多毛番茄和普通番茄中差异表达，且多毛番茄ShNAC4与普通番茄SlNAC4在氨基酸序列上差异较大。初步分析表明，超量表达ShNAC4转基因植株矮化，难直立，对低温更敏感。共表达分析表明NAC4同果胶甲酯酶及ACC合成酶具有相似的表达模式。我们推测NAC4可能通过调控细胞壁发育和/或乙烯生物合成负调控番茄的抗寒性。为进一步阐明NAC4在番茄生长发育、低温应答中的功能及其调控机制，揭示ShNAC4和SlNAC4序列的差异是否会导致二者分子及生物学功能的不同，本项目将对ShNAC4和SlNAC4的表达模式、亚细胞定位、转录激活活性、在低温应答、细胞壁发育、乙烯生物合成中的功能及调控机制进行比较分析，并在此基础上，进一步探究细胞壁发育、乙烯生物合成与植株抗寒性之间的关系。

前期, 我们发现低温胁迫下NAC转录因子NAC4在多毛番茄和普通番茄中差异表达。本项目分别从多毛番茄和普通番茄克隆了ShNAC4和SlNAC4。二者编码蛋白均含NAC保守结构域，序列相似性96.5%，有14个氨基酸位点发生变异。组织表达谱分析表明，ShNAC4和SlNAC4在多毛番茄和普通番茄各组织中的表达模式相似，均以在老叶中的表达量最高，花其次。ShNAC4和SlNAC4均受低温、干旱和高盐胁迫诱导表达，且在胁迫处理后期，SlNAC4的表达显著高于ShNAC4。亚细胞定位和转录激活活性分析表明，ShNAC4和SlNAC4均定位于细胞核，都没有转录激活活性。为解析NAC4的功能，我们构建了ShNAC4超量、SlNAC4超量和RNAi植物表达载体，并获得转基因植株。同野生型相比，ShNAC4转基因株系植株矮化，叶片失水速率及衰老加快，对低温、干旱和高盐等非生物胁迫的抗性降低。采用RNA-Seq技术对正常及低温胁迫下，转基因株系和野生型间基因表达差异进行了分析，鉴定了一批受ShNAC4调控的下游基因。通过分析发现，转基因株系与野生型间差异表达基因主要富集在“代谢”、“细胞进程”和“胁迫应答”等生物学进程。在胁迫应答相关差异表达基因中，绝大多数基因的表达被抑制。这些基因中有些已被证实正调控植物抗逆性，如Solyc07g014690.2.1、 Solyc07g040960.1.1、Solyc07g005210.2.1等。通过分析发现，乙烯生物合成途径ACS （Solyc02g063540.1.1）和ACO （Solyc04g009860.2.1、Solyc11g010400.1.1及Solyc09g089780.2.1）基因在转基因株系中上调表达，而乙烯信号转导途径ERF基因（Solyc04g012050.2.1、Solyc11g042560.1.1、Solyc12g009240.1.1、Solyc10g050970.1.1等） 在转基因株系中下调表达。以上结果表明，ShNAC4通过上调乙烯生物合成途径基因、抑制乙烯信号转导途径ERFs及其他非生物胁迫应答基因表达负调控番茄非生物胁迫抗性。本研究初步阐明了ShNAC4负调控番茄非生物胁迫抗性的分子机制，为番茄非生物抗性遗传改良提供了重要候选基因，为进一步解析番茄非生物胁迫应答的分子调控网络奠定了基础。