花生iso-Ara h3基因在种子抗黄曲霉中的功能研究

Aflatoxiin contamination of peanut (Arachis hypogaea. L) seeds is one of the most severe food safety factors that impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. The development of host-plant resistance would be an effective approach to eliminate aflatoxin accumulation in peanut. Unfortunately, the progress in transferring the resistance genes from these resistant lines into commercial cultivars has been slow, due to lack of understanding of the resistance mechanism and markers associated with resistance. In this study, a peanut seed iso-Ara h3 gene, which had been identified to associate with drought stress and Aspergillus infection in our previous study, was cloned. And than iso-Ara h3 was inserted into pET28a vecter to obtain recombinant protein and study the enzyme activity and in vitro function in resistance to Aspergillus flavus infection. To identify the in vivo-function of iso-Ara h3 in peanut and Arabidopsis in responding to drought and Aspergillus flavus stresses, this gene was cloned into pCAMBIA1301 vecter and than transformed into peanut or Arabidopsis. And some resistance associated genes in the transgenic plants were detected too. Moreover, a comprehensive analysis of promoter of iso-Ara h3 was performed to display signal regulation in responding to Aspergillus flavus infection. In a word, this study would be help for breeding of aflatoxin-resistance peanut and detection of food safety.

花生的黄曲霉毒素污染是限制花生产业发展和危害人类食品安全的重要因素。由于花生抗黄曲霉分子遗传机制不明，目前国内外花生抗性育种一直进展缓慢。培育耐旱和抗黄曲霉品种是最有效、经济的解决措施。本项目拟以前期花生蛋白质组学研究为基础，克隆与干旱胁迫、黄曲霉侵染相关的花生种子基因iso Ara h3。通过原核表达鉴定iso Ara h3基因的酶活性及体外抑菌功能。通过构建植物超表达载体和反义表达载体，转化花生和拟南芥，对转基因植株进行相关的基因表达和抗性分析，明确iso Ara h3在花生抗黄曲霉和耐旱中的功能。通过对iso Ara h3基因的上游顺式作用因子进行系统研究，明确其调控途径，并在此基础上结合生物信息学方法建立以重要基因为主线的花生抗黄曲霉调控网络加权矩阵模型。本项目的实施对于阐述花生抗黄曲霉调控途径、解决花生黄曲霉污染和

深入研究花生抗黄曲霉分子机制是促进花生抗性育种的关键。本研究通过PCR克隆获得花生iso-Arah3基因组全长, 其有5个外显子，蛋白序列与花生致敏原3和胰蛋白酶抑制因子同源性极高，N端有40个氨基酸的抗菌肽。.通过qRT-PCR分析iso-Arah3 时空、诱导表达模式，发现iso-Arah3在根、种子发育和萌发过程中有重要表达功能，在种子和根中显著受干旱胁迫和黄曲霉诱导调节。表明iso-Arah3与种子成熟和响应干旱及黄曲霉侵染有相关性。通过瞬时表达试验分析表明iso-Arah3基因定位于细胞质。.利用原核超表达系统制备纯度84.5%的iso-Arah3融合蛋白和抗菌肽，经复性，进行体外抑菌分析，发现抗菌肽体外抑菌效果明显好于iso-Arah3蛋白，说明抗菌肽是iso-Arah3抗菌活性的功能核心区。利用纯化的融合蛋白免疫兔子，制备iso-Arah3特异、高效价多抗血清。利用多抗对各组织内源iso-Arah3蛋白进行Western-Blot检测，发现iso-Arah3蛋白在种子、胚、根中有大量积累，而在种子、胚、根和茎中有大量TI蛋白积累，推测花生iso-Arah3蛋白是TI的前体蛋白，在生理调节下，iso-Arah3被各种酶类修饰产生有活性TI。.通过农杆菌侵染拟南芥，获得4个纯合转iso-Arah3拟南芥株系，对转iso-Arah3拟南芥的生理生化分析，发现转iso-Arah3拟南芥的叶片H2O2含量明显增高；在干旱条件下，转iso-Arah3拟南芥株系1、2和3的根系明显好于野生型和阳性对照。明确了iso-Arah3基因的异源超表达功能及抗逆/病的作用机制。.通过基因组步移技术克隆iso-Arah3基因1228bp启动子（I3P），I3P含有大量响应干旱、病害、氧胁迫、光调控和激素的顺式元件。将I3P缺失片段与报告基因GUS组成融合基因，经农杆菌转化拟南芥，经GUS化学染色，发现I3P0驱动GUS在中胚轴和叶节处大量表达，I3P1和2驱动GUS在中胚轴、茎和叶节中表达。I3P3在侧根中有少量表达。推测I3P的-523~-916区是调控中胚轴、叶节和茎特异表达的关键区域，活性受各种病害（包括黄曲霉菌的侵染）及光信号的调控。.总之，本研究深入分析了iso-Arah3及其启动子的抗病活性和功能机制，为加快抗病花生品种的培育和解决花生的黄曲霉毒素污染问题奠定基础。