摘要 【目的】探究舞毒蛾取食对小黑杨苯丙氨酸代谢途径中关键酶诱导防御的影响,为明确杨树抗虫性分子机制提供理论依据,并为利用基因工程手段提高寄主抗虫性提供基因材料。【方法】通过RT-PCR法获得小黑杨C4H和4CL基因全长序列,并进行基因特性分析;采用邻接法构建C4H和4CL系统进化树;利用分光光度计法比较舞毒蛾取食和机械损伤处理下小黑杨C4H和4CL活性,并运用实时荧光定量RT-PCR技术测定2种损伤处理对C4H和4CL基因表达量的影响。【结果】从小黑杨转组文库中克隆鉴定获得2个属于p450家族的C4H基因(命名为C4H-1和C4H-2)以及1个多结构域蛋白超家族(AFD_class_I)4CL基因,全长基因开放阅读框大小为1518~1740 bp,编码505~579个氨基酸,蛋白质分子质量为58.00~63.03 kDa,理论等电点pI为5.63~9.09。系统进化树分析表明,小黑杨C4H-1与银白杨亲缘关系近聚为一类;而C4H-2与毛白杨、欧洲山杨、毛果杨×美洲黑杨和毛果杨具有较高同源性聚为一类,小黑杨4CL与毛果杨×美洲黑杨和毛果杨亲缘关系较近。舞毒蛾取食诱导小黑杨C4H-1、C4H-2和4CL基因表达量增加,分别为对照组的6.86、1.15和1.50倍;而机械损伤诱导C4H-1基因表达上调,抑制C4H-2和4CL基因表达下调,分别为对照组的1.80、0.71和0.60倍。取食和机械损伤均能诱导小黑杨C4H和4CL活性增加,且取食诱导显著高于机械损伤。【结论】舞毒蛾取食显著诱导小黑杨C4H和4CL活性增加,可能分别来源于C4H-1、C4H-2和4CL基因表达量增加。舞毒蛾取食参与诱导小黑杨苯丙氨酸生物合成途径防御反应,可为进一步明确小黑杨诱导抗性机制以及探讨小黑杨木质素合成防御昆虫取食提供基础理论。 【Objective】Phenylalanine is a precursor of many plant compounds that are essential for plant reproduction,growth,development,and defense against different types of stress.Phenylalanine biosynthesis pathway is one of the most important secondary metabolic pathways in plants.In the phenylalanine biosynthesis pathway,the cinnamate 4-hydroxylase(C4H)and 4-coumarate CoA ligase(4CL)are involved in the core reaction of phenylpropane metabolism.Lymantria dispar is a major forest pest in the world.In this study,our objective is to explore the effects of Lymantria dispar herbivore on key enzymes in phenylalanine biosynthesis pathway of Populus simonii×P.nigra for inducing defense,and to provide genetic materials for promoting host resistance by genetic engineering method.【Method】The full length of C4H and 4CL was cloned by RT-PCR technology and its characteristics were analyzed in this study.The phylogenetic trees of C4H and 4CL were constructed by Neighbor-joining method,respectively.The activities of C4H and 4CL were compared by using spectrophotometry under the treatment of Lymantria dispar herbivore and mechanical wounding.The expression of C4H and 4CL genes under the two wounding treatments was determined by real-time RT-PCR technology.【Result】Two C4H genes(namely C4H-1 and C4H-2)belonging to p450 family and one 4CL from multi-domain protein superfamily(AFD_class_I)were cloned and identified from the transcriptome of Populus simonii×P.nigra.The open reading frames of the full-length genes were 1518-1740 bp,encoding 505-579 amino acid residues with the molecular mass of 58.00-63.03 kD and theoretical pI of 5.63-9.09.Phylogenetic analysis showed that the C4H-1 of Populus simonii×P.nigra was clustered into a group with that of P.alba.The C4H-2 of Populus simonii×P.nigra was clustered into a group with that of P.tomentosa,P.tremuloides,P.trichocarpa×P.deltoides and P.trichocarpa.The 4CL of Populus simonii×P.nigra is closely related to that of P.trichocarpa×P.deltoids and P.trichocarpa.The results showed
出处 《林业科学》 EI CAS CSCD 北大核心 2021年第3期108-116,共9页 Scientia Silvae Sinicae
基金 “十三五”国家重点研发计划项目(2018YFC1200400) 东北林业大学本科生创新项目(201810225109)。
关键词 舞毒蛾 取食 小黑杨 肉桂酸-4-羟化酶 4-香豆酸CoA连接酶 机械损伤 基因表达 Lymantria dispar herbivore Populus simonii×P.nigra cinnamate 4-hydroxylase 4-coumarate CoA ligase mechanical wounding gene expression
分类号 S718.7 [农业科学—林学]
