调节抗低温糖化马铃薯转化酶抑制子StInvInh2关键转录因子的克隆及其功能研究

Cold-induced sweetening (CIS) of tubers is a major trail affecting potato processing quality. Our results indicated that activity of acid invertase StvacINV1 was a key factor regulating the CIS of potato tubers. In addition, StInvInh2 as an inhibitor StvacINV1 specifically capped StvacINV1 activity, which is an important way to regulate StvacINV1 activity. StvacINV1 activity was inhibited due to high induced mRNA abundance of StInvInh2 in cold-stored potato tubers with CIS-resistance. The promoter activity of StInvInh2 was proved to be increased in cold-stored potato tubers with CIS-resistance. However, the regulatory mechanism of StInvInh2 is still unclear in CIS-resistant potato tubers. In present project, the candidate transcript factors regulating StInvInh2 will be screened by yeast one hybrid and bioinformative approaches. Combined with the analysis of interacted cis-element(s) and protein(s), the key transcript factors regulating StInvInh2will be identified by the transgenetic approach. The results will deepen our understanding in the molecular mechanism of CIS and will provide novel strategies and the gene recourses for improving potato processing quality.

马铃薯低温糖化是影响加工品质的主要性状。前期研究表明，酸性转化酶StvacINV1活性是调节低温糖化的主要因子，转化酶抑制子StInvInh2与StvacINV1互作并抑制其活性，是调节StvacINV1活性的重要途径。在抗低温糖化马铃薯中，低温贮藏块茎诱导表达StInvInh2是抑制StvacINV1活性升高的主要原因，且StInvInh2启动子具有低温诱导表达活性，但其调节机制仍然不清楚。本研究拟通过酵母单杂交结合生物信息学途径筛选调节StInvInh2的候选转录因子，结合EMSA实验验证StInvInh2启动子互作蛋白，进一步瞬时表达体系鉴定调节StInvInh2的关键转录因子，明确调节StInvInh2的关键转录因子。本研究结果将补充和完善抗低温糖化分子机制，为马铃薯加工品质改良提供新基因资源和新途径。

马铃薯转化酶抑制子StInvInh2通过与StvacINV1互作并抑制其活性，是调节低温糖化的关键因子。低温贮藏块茎升高StInvInh2的表达是导致抗低温糖化的重要原因，然而其调节机制不清楚。本研究主要克隆马铃薯低温糖化抗/感材料中StInvInh2的启动子序列，分析了其中的顺式作用元件，通过遗传转化研究其功能，结果发现StInvInh2启动子在低温糖化抗性和敏感两种基因型中都在块茎中优势表达，低温贮藏块茎中低温糖化抗性基因型中StInvInh2启动子活性升高，但在低温糖化敏感基因型中下降，说明低温糖化抗感基因型中StInvInh2的启动子活性发生分化，而这种分化的原因更可能与上游调节其表达的转录因子的活性状态更相关。基于低温糖化抗、感基因型材料块茎低温贮藏期的表达谱数据，通过通过共表达分析结合StInvInh2启动子顺式元件可能结合的转录因子类型，初步筛选出与StInvInh2表达相关的6个候选转录因子。进一步通过在烟草中的双荧光瞬时激活实验表明，其中2个ERF类的转录因子具有激活StInvInh2启动子活性能力。另外，通过EMSA实验证实2个ERF类的转录因子能特异的与StInvInh2启动子中的LTRE元件结合。这些结果暗示2个ERF类的转录因子可能是调节StInvInh2的转录的关键转录因子，他们主要通过特异的与StInvInh2启动子中的LTRE元件结合并激活其活性。整个研究结果为明确StInvInh2在低温糖化抗、感材料中的转录调节机制提供新的线索，加深和完善对抗低温糖化分子机制的理解，为抗低温糖化育种提供新资源和新思路。