摘要：Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop ***-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic ***,enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental *** open reading frames(uORFs)are translational control elements located in the 5′untranslated region(UTR)of eukaryotic mRNAs and may repress the translation of downstream *** investigate the function of three uORFs from the 5′-UTR of ACCELERATED CELL 11(uORFsACD11),we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene *** or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different ***,uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant *** coupled with the PATHOGENESIS-RELATED GENE 1(PR1)promoter,the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2(AtLecRKVI.2)during pathogen attack and enhance plant resistance to Phytophthora *** findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack,while for ensuring lower levels of protein expression at normal conditions.

摘要：为建立强雄腐霉Pythium arrhenomanes的快速分子检测技术,基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)以β-tubulin基因为靶标序列,设计强雄腐霉的特异性引物,建立了一种准确快速的LAMP检测方法,并对该检测方法的特异性、灵敏度和实际应用效果进行评估。25 μL LAMP最终反应体系为:10×ThermoPol Buffer 2.5 μL、10 mmol/L dNTPs 3.5 μL、6 mmol/L MgSO4 2 μL、5 mol/L甜菜碱4 μL、40 μmol/L FIP-1/BIP-1各1 μL、5 μmol/L F3-1和B3-1各1 μL、40 μmol/L LB-1 1 μL、Bst DNA polymerase 1 μL、2.5 mmol/L HNB 1.9 μL、模板DNA 2 μL、灭菌水3.1 μL。LAMP体系在等温64℃条件下反应60 min,HNB显色反应显示天蓝色即为阳性反应;扩增产物经2%琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性检测结果显示,LAMP体系能特异性地检测出强雄腐霉,而其它卵菌近缘种和常见植物病原真菌均未检测出。灵敏度检测结果显示,LAMP体系的最低检测灵敏度为10 pg/μL,是普通PCR反应灵敏度的1 000倍。在实际应用中,LAMP体系能够快速检测出人工接种和实际发病玉米组织中的病原菌。表明本研究建立的快速、准确、可视化的LAMP检测方法能在60 min内检测出强雄腐霉。

摘要：[目的]建立一种快速、准确的检测瓜果腐霉菌(Pythium aphanidermatum)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,为诊断和防治瓜果腐霉菌引起的植物病害提供技术支撑。[方法]通过比较基因组学方法,在瓜果腐霉菌基因组上筛选有序列特异性的基因作为检测的靶标基因,设计LAMP特异性引物,建立一种基于颜色判定的快速、准确的LAMP检测方法,随后对该检测方法的特异性、灵敏度和发病组织实际检测等进行分析评价。[结果]鉴定到1个编码Trypsin蛋白酶的基因是瓜果腐霉菌特异的基因,以该序列作为靶标建立了LAMP检测方法。在扩增前加入染料羟基萘酚蓝(HNB)作为指示剂,在等温条件(64℃)下进行核酸扩增反应60 min,即可通过肉眼直接观察结果,HNB显示天蓝色即为阳性反应;同时,扩增产物经琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性试验中,建立的LAMP检测方法能将瓜果腐霉菌和其他病原菌区分开。灵敏度试验中,LAMP检测方法的最低检测限为100 pg·μL-1,与Real-time PCR反应灵敏度一样,是普通PCR反应灵敏度的100倍。发病组织实际检测试验中,该LAMP方法能检测出人工接种发病植株中的瓜果腐霉菌。[结论]成功建立了一种适用于基层使用的针对瓜果腐霉菌的快速检测技术,为该菌所致病害的准确诊断奠定了技术基础。