铝影响甜高粱β-1,3葡聚糖酶I（SbGlu1）活性的分子机制及其遗传改良研究

Aluminum (Al) can induce rapid deposition of callose in plant root apex. However, the regulatory mechanism of Al on callose deposition is still unclear. Previously study showed that under Al stress, the transcriptional expression level of β-1,3 glucanase I (SbGlu1) increased in sweet sorghum. However, SbGlu1 activity decreased obviously. Further research has been made from the perspective of enzyme activity by applicant. We found that Al3+ may directly bind to the amino acid nearby the catalytic site of SbGlu1, which finally inhibiting the catalytic activity of SbGlu1 for degrading callose. Therefore, we firstly proposed to verify the binding mode and sites of Al on SbGlu1, thereby elucidating the molecular mechanism involved in the effect of Al on the activity of SbGlu1. Secondly, systematically screen will be made to identify one or more Al tolerance SbGlu1M mutants, whose activity can not be affected by Al but keep the catalytic activity of wild type SbGlu1. We further use the promoter of SbGlu1 to regulate the transcriptional express of SbGlu1M in order to obtain the transgenic plants with higher capacity for Al tolerance and effectively avoiding the gene silence. The study further deepen the understanding for us of Al induced root callose accumulation, which is one of most important Al toxicity mechanisms, and also, provides new application of SbGlu1 or other Al resistant gene in molecular genetic improvement.

铝（Al）能诱导植物根尖快速积累胼胝质，但Al对胼胝质形成的调控机制尚不清楚。前期研究表明，Al胁迫下，甜高粱β-1,3葡聚糖酶I（SbGlu1）转录水平上调，然而其酶活性却明显下降。申报人近期在蛋白水平上的研究发现，Al3+可能直接通过离子键结合于SbGlu1的活性位点附近，进而抑制SbGlu1的催化活性，影响胼胝质降解。因此，本研究拟首先验证Al在SbGlu1上的结合方式和结合位点，阐明Al影响SbGlu1酶活性的分子机制；其次，通过改造SbGlu1，系统地筛选获得不受Al影响且与野生型SbGlu1酶活性相当的SbGlu1M突变体（即“耐Al型SbGlu1M”）；最终利用SbGlu1自身启动子，获得有效规避基因沉默且高效耐Al的转基因材料。该研究可深化对Al诱导根尖胼胝质积累这一Al毒害机制的认识，亦可为SbGlu1或其它耐Al基因在分子遗传改良中的应用开辟新的思路。

铝（Al）毒害是酸性土壤上限制农作物产量的主要因素。铝能诱导植物根尖快速积累胼胝质，进而阻碍胞间连丝并影响共质体运输。甜高粱SbGlu1编码一个β-1,3-葡聚糖酶，该酶催化胼胝质降解，因此在甜高粱Al胁迫应答中发挥重要作用。然而Al调控SbGlu1的具体机制，包括对酶性质和酶数量的调控尚不清楚，因此本研究首先分析Al直接结合于SbGlu1蛋白，进而影响SbGlu1酶催化活性的分子机制；其次解析了Al胁迫下SbGlu1转录水平的调控机制。主要研究内容和重要结果如下：.1. 甜高粱SbGlu1蛋白等电点为4.96，是酸性蛋白质；SbGlu1与大麦HvGlu1高度同源，其C端具备糖基水解酶17家族结构域。.2. 酶活性分析表明，Al直接抑制SbGlu1的催化活性。.3. 根据SbGlu1的蛋白三维结构模型，从电性和空间两个角度，推测Al3+可能通过离子键结合于SbGlu1的活性位点附近，即由至少三个酸性氨基酸（谷氨酸E99，E240和E297）构成的酸性口袋，从而抑制SbGlu1的催化活性。.4. 将SbGlu1上三个谷氨酸分别定点突变为天冬氨酸（D）、丝氨酸（S）和丙氨酸（A），并进行突变体蛋白体外表达与纯化；酶活性分析表明，三个谷氨酸均参与和Al3+的直接结合。.5. 完成SbGlu1及SbGlu1突变体植物表达载体构建与遗传转化，可用于耐Al型SbGlu1突变体的功能验证。.6. 鉴定了甜高粱转录因子SbSTOP1d的耐Al功能，并发现SbSTOP1d正向调控SbGlu1的转录。.7. 验证了SbSTOP1d与SbGlu1启动子的直接结合，揭示了Al通过转录因子SbSTOP1d，调控SbGlu1的转录水平进而影响酶数量。.该研究可深化对Al诱导根尖胼胝质积累这一Al毒害机制的认识，亦可为SbGlu1或其它耐Al基因在分子遗传改良中的应用开辟新的思路。.