磷脂酶Dα1在TuMV激活叶绿素降解相关基因表达中的功能分析  

The role of phospholipase Dα1 in the expression of chlorophyll degradation related genes activated by TuMV

作　　者：林佳宇 张世杰 徐传涛[3] 杜林林 孙炳剑[1] 孙航军 LIN Jiayu;ZHANG Shijie;XU Chuantao;DU Linlin;SUN Bingjian;SUN Hangjun(College of Plant Protection,Henan Agricultural University,Zhengzhou 450046,China;Baoji Tobacco Company of Shaanxi Province,Baoji 721000,China;Luzhou Tobacco Company of Sichuan Province,Luzhou 646000,China)

机构地区：[1]河南农业大学植物保护学院,河南郑州450046 [2]陕西省烟草公司宝鸡市公司,陕西宝鸡721000 [3]四川省烟草公司泸州市公司,四川泸州646000

出　　处：《河南农业大学学报》2024年第5期783-790,共8页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(31901855);中国烟草总公司陕西省公司科技项目(KJ-2023-05)。

摘　　要：【目的】研究磷脂酶Dα1(phospholipase Dα1)及其产物磷脂酸(phosphatidic acid,PA)在芜菁花叶病毒(turnip mosaic virus,TuMV)促进植物叶绿素降解相关基因表达过程中的作用。【方法】通过UV-2800紫外分光光度计检测TuMV对本氏烟叶绿素含量的影响。利用Real-time qPCR检测TuMV侵染或瞬时表达6K2蛋白对本氏烟叶绿素降解相关基因NbNYE1(Nicotiana benthamiana NON-YELLOWING1)、NbNYE2(Nicotiana benthamiana NON-YELLOWING2)和NbPAO(Nicotiana benthamiana pheide a oxygense)mRNA相对表达水平的影响。敲除NbPLDα1(Nicotiana benthamiana phospholipase D alpha 1)或正丁醇降低磷脂酶D产生的磷脂酸含量,检测对TuMV诱导的NbNYE1、NbNYE2和NbPAO mRNA相对表达水平的影响。体外喷施磷脂酸对NbNYE1、NbNYE2和NbPAO mRNA相对表达水平的影响。【结果】TuMV侵染导致野生型本氏烟叶片叶绿素含量下降48%。TuMV侵染的野生型本氏烟中NbNYE1、NbNYE2和NbPAO表达量分别为健康野生型本氏烟的7.61、15.80和5.19倍。TuMV侵染的NbPLDα1敲除突变体本氏烟中NbNYE1、NbNYE2和NbPAO表达量分别为健康野生型本氏烟的2.35、7.83和1.29倍。正丁醇处理后,TuMV侵染的野生型本氏烟NbNYE1、NbNYE2和NbPAO表达量与健康野生型本氏烟无明显差异。喷施磷脂酸的叶片中NbNYE1、NbNYE2和NbPAO表达量分别为清水处理野生型本氏烟的12.39、3.64和8.26倍。此外,瞬时表达6K2蛋白叶片中NbNYE1、NbNYE2和NbPAO表达量分别为瞬时表达GFP野生型本氏烟的5.64、4.67和3.45倍。【结论】TuMV促进叶绿素降解相关基因表达依赖磷脂酶Dα1及其产物磷脂酸,病毒编码的6K2蛋白是促进叶绿素降解相关基因的关键致病因子。【Objective】Investigating the role of phospholipase Dα1(PLDα1)and its product phosphatidic acid(PA)in the expression of turnip mosaic virus(TuMV)-induced chlorophyll degradation related genes in plants.【Method】The impact of TuMV on chlorophyll content in Nicotiana benthamiana was assessed using a UV-2800 UV spectrophotometer.Real-time qPCR was employed to analyze the effect of TuMV infection or transient expression of the 6K2 protein on the mRNA relative expression levels of chlorophyll degradation related genes Nicotiana benthamiana NON-YELLOWING1(NbNYE1),Nicotiana benthamiana NON-YELLOWING2(NbNYE2)and Nicotiana benthamiana pheide a oxygense(NbPAO)in N.benthamiana.Knockout of Nicotiana benthamiana phospholipase D alpha 1(NbPLDα1)or reduction of PA levels generated by PLD using 1-butanol was conducted to investigate the influence on TuMV-induced changes in mRNA relative expression levels of NbNYE1,NbNYE2,and NbPAO.Exogenous application of PA was used to evaluate its effect on the mRNA relative expression levels of NbNYE1,NbNYE2,and NbPAO.【Result】TuMV infection resulted in a 48%reduction of chlorophyll content in wild-type N.benthamiana leaves,with mRNA expression levels of NbNYE1,NbNYE2,and NbPAO increasing by 7.61,15.80 and 5.19-fold of those in healthy wild-type N.benthamiana,respectively.The expression levels of NbNYE1,NbNYE2,and NbPAO in TuMV-infected NbPLDα1 knockout mutant N.benthamiana were 2.35,7.93 and 1.29-fold higher than those of healthy wild-type N.benthamiana,respectively.After 1-butanol treatment,there was no significant difference in expression levels of NbNYE1,NbNYE2,and NbPAO between TuMV-infected wild-type N.benthamiana and healthy plants.The expression levels of NbNYE1,NbNYE2,and NbPAO in wild-type N.benthamiana sprayed with PA were 12.39,3.64 and 8.26-fold higher than those of plants treated with water,respectively.In addition,the expression levels of NbNYE1,NbNYE2,and NbPAO by transient expression of the 6K2 protein were 5.64,4.67 and 3.45-fold higher than those of expressing

关 键 词：芜菁花叶病毒 本氏烟 叶绿素降解 6K2蛋白 磷脂酸 磷脂酶D 

分 类 号：S432.1[农业科学—植物病理学]