猕猴桃淀粉酶基因BAM响应低温的分子机制研究

The freezing injury, which is one of the most important abiotic stresses in horticultural crops, can influences the production area of kiwifruit(Actinidia Lindl.), which results in a dramatically reduction or destroying the garden, such a problem needs to be urgently solved. Breeding fine cultivar with strong cold tolerance is one of the important target in kiwifruit breeding. In this project, Actinidia arguta with a high cold-resistant will be used as experiment material, β-amylase (BAM) gene, which was identified had a significant expression in the extrem low temperature in the previous study, will be used as a mainly target gene. qRT-PCR will be used to analysis the expression during different low temperature, trying to learn the relationship between the expression and the kiwifruit cold resistance. The activity of β-amylase, content of soluble sugar and other physiology parameters will be detected to analysis the BAM gene function during kiwifruit cold resistance. Subcellular localization will be used to locate the BAM gene. Transgene and RNAi will be used in Arabidopsis and A. arguta to verify the BAM gene function. The fulfill of this will provide theoretical basis for the study of molecular mechanism of cold resistance on kiwifruit, giving us a clearly regulatory network for kiwifruit responding to low temperature.

低温作为园艺作物中重要的非生物胁迫之一，直接影响到猕猴桃种植，轻者造成减产，重者导致死树毁园，培育抗寒性强的优良猕猴桃品种已经成为育种的重要方向之一。本研究以抗寒性极强的软枣猕猴桃为材料，以前期试验挖掘的在低温下显著表达的β淀粉酶（BAM）基因为入手点，利用实时荧光定量技术研究BAM基因在不同低温下的表达特性，了解BAM基因表达对猕猴桃抗寒性的影响，结合β淀粉酶活性、可溶性糖含量等生理指标验证BAM基因参与猕猴桃抗寒性过程，利用亚细胞定位进行BAM基因定位，通过转基因和RNAi技术在拟南芥和软枣猕猴桃中进行功能验证，并采用酵母单杂交技术分析和BAM基因相互作用的调控基因，通过综合分析，明晰以BAM为主线的软枣猕猴桃应对低温的整个调控网络，为猕猴桃抗寒性分子机制研究提供理论基础。

低温作为园艺作物中重要的非生物胁迫之一，直接影响到猕猴桃种植，轻者造成减产，重者导致死树毁园。前期研究显示，猕猴桃β淀粉酶（BAM）基因能够在低温下显著表达。因此本项目以BAM基因为入手点，以抗寒性极强的软枣猕猴桃为材料，利用实时荧光定量技术研究BAM基因在不同低温下的表达特性，利用亚细胞定位进行BAM基因定位，通过转基因在拟南芥和猕猴桃中进行功能验证，并采用酵母单杂交技术分析和BAM基因相互作用的调控基因。通过研究，我们克隆到BAM基因并命名为AaBAM3.1基因，其受低温诱导，AaBAM3.1基因在休眠期枝条中的表达显著高于生长期，在叶片中的表达量高于根、茎；它定位于叶绿体中；转基因植株在低温下的可溶性糖含量显著提高，PRO、MDA、CAT含量和SOD活性在转基因植株中明显升高，转基因植株的相对电导率降低即抗寒性提高，在拟南芥转基因也得到相似的结果，证明了AaBAM3.1在猕猴桃中的抗寒功能。通过AaBAM3.1启动子元件分析发现启动子中包含CBF结合元件、JA结合元件以及ABA结合元件等，验证了AaBAM3.1启动子的转录激活活性，通过采用酵母单杂交筛选AaBAM3.1基因与AaCBF4互作，并进一步进行双荧光素酶验证试验验证。项目的开展猕猴桃抗寒性分子机制研究提供理论基础。