苹果6-磷酸山梨醇脱氢酶基因启动子的功能分析与应用

Sorbitol is the primary photosynthetic product in many species of Rosaceae. In addition, sorbitol is an efficient osmolyte. Its accumulation can improve plant tolerance against abiotic stress.NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH)play a key role in the biosynthesis of sorbitol.In the previous study, we found that the expression of S6PDH promoter (S6PDHp)may be tissue-specific and stress-inducible. In order to find out the functional characteristics of S6PDHp and further put it into use,in this study,the 5`deletion of S6PDHp will be transformated to tomato and Arabidopsis to analysis promoter characteristics by the expression of GUS and find key regions in the promoter.The transcription initiation sites for apple S6PDH expressed in different conditions will be mapped by RLM-5`RACE.The activity of S6PDHp will be increased by adding the enhancer derived from other promoters.Through this project implementation,we can not only explore the characteristics of S6PDHp and improve our understanding on expression regulation of S6PDH,but also make use of S6PDHp as a kind of tissue-specific and stress-inducible promoter to the genetic transformation of plants.

山梨醇是大多数蔷薇科植物主要的光合产物。山梨醇也能作为小分子渗调物质参与植物对逆境的抵御反应。6-磷酸山梨醇脱氢酶(S6PDH)是这些植物体内生成山梨醇的关键酶。前期的研究结果表明，S6PDH基因的启动子（S6PDHp）可能具有组织特异表达和胁迫诱导表达等特性。为探明S6PDHp的功能特性并加以利用，本项目拟通过将不同长度启动子缺失体转化番茄和拟南芥，通过GUS基因的表达来分析S6PDHp的组织特异表达和逆境诱导表达等特性，寻找关键的顺式作用调控区域；通过RLM-5`RACE法发现不同条件下S6PDH基因的转录起始位点；参考其他启动子的研究，通过增加一些增强子来提高S6PDHp的活性。通过该项目的实施，不仅可以探明S6PDHp的功能特性，加深对S6PDH基因表达调控的认识，还可以将S6PDHp作为一个组织特异表达和胁迫诱导表达型启动子广泛的应用于不同植物的遗传转化研究中。

启动子的功能特性是基因表达调控的重要一环。因此，为探明山梨醇合成关键酶6-磷酸山梨醇脱氢酶基因启动子（S6PDHp）的功能特性并加以利用，本项目通过S6PDHp系列缺失体载体构建和转化等方法对其进行了研究。研究结果如下：（1）设计引物克隆S6PDHp5’端的4个缺失体，然后构.建S6.PDHp缺.失体与GUS基.因融.合的稳.定表.达载.体，通.过农杆菌介导法转.化拟南.芥。对获得的转基因植株进行ABA和4℃低温处理，通过不同时间点样品中GUS蛋白活性来分析S6PDHp的逆境响应特性。研究结果发现-836部分具有最高的低温响应能力。在A.BA处.理下，-1396和-836在胁迫初期和中期响应最为明显。（2）设计引物克隆S6PDHp5’端的6个缺失体，然后构.建S6.PDHp缺.失体与GUS基.因融.合的瞬时表.达载.体，通.过农杆菌介导法转.化烟草叶片。通.过对瞬.时转.化后的烟.草叶片中的GUS活.性分.析研究启动子特性。结果发.现所克.隆的6.个启.动子缺.失体都.是具.有活.性的。对瞬时转化的烟草进行了不同条件的处理后发现，-2369、-2122、-1719和-646对水杨酸处理响应明显；-2369、-1066和-646对低温处理响应明显；-1066和-251对ABA处理响应明显；只有-2369对黑暗处理响应明显；-2369、-1066和-646对赤霉素处理响应明显；-2122、-1066和-251对茉莉酸甲酯处理响应明显。（3）将S6PDHp-GU.S融.合表.达载.体转.化番.茄，并对转基.因番.茄阳.性植.株的各.个组.织器.官进.行GU.S化.学染.色和GU.S蛋.白活.性分.析，发.现该启.动子在除.了根.部以.外的其.他组.织器.官中均.有不.同程.度的活.性。（4）通过RACE发现，S6PDHp在正常生长和逆境下具有相同的转录起始位点。（5）通过构建表达载体时在启动子和GUS基因之间加入Omega序列，可以调高启动子活性。（6）通过酵母单杂交试验，发现了23号和90号基因可能与S6PDHp结合。经过酵母回转和EMSA进一步验证，发现只有90号基因可以与S6PDHp结合。以上研究初步探明了S6PDHp的一些功能特性，加深了对S6PDH基因表达调控的认识，还为S6PDHp应用于植物遗传转化研究奠定了基础。