猕猴桃NPR1类似基因家族成员的克隆与抗病功能解析

Kiwifruit diseases were increased with the rapid expansion of cultivated area for the past few years, especially bacterial canker disease of kiwifruit. Bacterial canker disease of kiwifruit is a serious danger to the production and development of kiwifruit. The world kiwifruit industry was caused serious economic losses by this disease. NPR1 or like gene was critical node in signal transduction pathways of plant disease resistance, plays a very important role in the process of plant resistance to fungal, bacterial, and viral diseases. The complete genome sequence of kiwifruit was sequenced in 2013. Bioinformatics analysis showed that kiwifruit contains 5 different NPR1 like gene sequences based on the complete genome sequence. cDNA sequences of all NPR1 family members will be cloned from the Jinkui which was a resistant varieties to bacterial canker disease. the function of NPR1 family members will be identified using the subcellular localization, expression pattern, disease resistance analysis and regulating the expression of downstream genes of transgenic Arabidopsis overexpressing these genes. The objective of this study was to figure out which member of five kiwifruit NPR1 gene family involved in disease resistance, and which signaling pathways involved in, and elucidate the molecular mechanism of kiwifruit NPR1 disease resistance gene family members. This study will provide new genetic resources for breeding disease resistance kiwifruit resource through the method of plant gene engineering, and has higher theoretical significance and application value.

近年来由于猕猴桃栽培面积迅速扩大，猕猴桃病害随之加剧，尤其是猕猴桃细菌性溃疡病，是一种毁灭性病害，给我国乃至全球猕猴桃产业造成了巨大的经济损失。NPR1及其类似基因是植物抗病信号转导途径中的关键节点，在抵抗真菌、细菌和病毒病的过程中起着非常重要的作用。猕猴桃全基因组序列已经测序完成，通过生物信息学分析得知该基因组中含有5个不同的NPR1类似基因。本项目以溃疡病抗病品种金魁为材料，利用RT-PCR技术克隆NPR1类似基因家族全部成员的cDNA序列，通过基因家族成员亚细胞定位、表达特性分析、转基因拟南芥过量表达的抗病性鉴定及其调控下游基因的表达等途径验证其功能特性。通过本项目的实施阐述猕猴桃5个NPR1类似基因家族哪些成员通过何种信号通路参与抗病性，阐明猕猴桃NPR1类似基因家族抗病成员的抗病分子机理，为利用植物基因工程增强猕猴桃属植物的抗病能力提供新的基因资源，具有较高的理论意义和应用价值。

猕猴桃细菌性溃疡病是一种严重威胁猕猴桃生产和发展的毁灭性病害，给猕猴桃产业造成了巨大的经济损失。NPR1类似基因是植物抗病信号转导途径中的关键节点，在抵抗真菌、细菌和病毒病的过程中起着非常重要的作用。本项目从红阳猕猴桃基因组数据库中获得5个NPR1类似基因家族成员，分别为Achn060941、Achn176311、Achn209911、Achn326501和Achn326871。利用RT-PCR技术从金魁猕猴桃中获得了3个AdNPR1类似基因和2个AdPR1类似基因，分别命名为AdNPR1.1、AdNPR1.2、AdNPR3、AdPR1.1、AdPR1.2。AdNPR1.1全编码区长为1749 bp，编码582个aa。AdNPR1.2全编码区长为1761 bp，编码586个aa。AdNPR3全编码区长为1770 bp，编码589个aa。AdPR1.1全编码区长为483bp，编码160个aa。AdPR1.2全编码区长为522 bp，编码173个aa。溃疡病病原菌可以显著诱导AdNPR3、AdPR1.1和AdPR1.2基因的表达；SA和MeJA处理均可以显著诱导AdNPR3、AdPR1.1和AdPR1.2基因的表达；ACC可以诱导AdNPR1.1、AdPR1.1 和AdPR1.2基因的表达。溃疡病病原菌和植物激素SA、MeJA和ACC未能诱导AdNPR1.2基因的表达。AdNPR1.1蛋白位于细胞核和细胞质中，AdNPR3蛋白位于细胞核中。将AdNPR1.1和AdNPR3基因分别构建植物表达载体，通过花粉管通道法转化拟南芥，获得了转基因株系。利用病原菌PstDC3000侵染转基因株系和非转基因株系，结果转基因株系表现出较强的抗性，转基因拟南芥中分别过量表达猕猴桃AdNPR1.1和AdNPR3可以诱导下游病程相关蛋白基因AtPR1 、AtPR2 和AtPR5基因的表达。AdNPR1.1可能参与乙烯信号转到通路；AdNPR3可能参与SA和MeJA信号转导通路。AdNPR1.2未参与这3种通路。AdNPR1.1和AdNPR3参与系统获得抗病性。AdNPR1.2可能和抗病性无关。本项目初步阐明了金魁猕猴桃AdNPR1 类似基因家族抗病基因成员的分子机理，为通过基因工程技术手段培育猕猴桃抗病性品种奠定了基础。