氮素亏缺对苹果愈伤组织硝态氮吸收及同化的影响  

Effects of nitrogen deficiency on nitrate uptake and assimilation of apple callus

作　　者：文滨滨 张新昊 沈红艳 武红玉 陈修德 肖伟 高东升 WEN Bin-bin;ZHANG Xin-hao;SHEN Hong-yan;WU Hong-yu;CHEN Xiu-de;XIAO Wei;GAO Dong-sheng(College of Horticulture Science and Engineering,Shandong Agricultural University/Shandong Collaborative Innovation Center for Fruit and Vegetable Production with High Quality and Efficiency/State Key Laboratory of Crop Biology,Tai'an,Shandong 271018,China)

机构地区：[1]山东农业大学园艺科学与工程学院/山东果蔬优质高效生产协同创新中心/作物学国家重点实验室

出　　处：《植物营养与肥料学报》2019年第11期1939-1948,共10页Journal of Plant Nutrition and Fertilizers

基　　金：山东省现代农业产业技术体系果品创新团队项目（SDAIT-06-01）

摘　　要：【目的】研究硝态氮亏缺对苹果叶片愈伤组织生长及硝态氮吸收同化的影响,了解苹果愈伤组织对硝态氮亏缺的响应机制,为进一步研究缺氮处理影响愈伤组织生长发育的分子机理提供理论依据。【方法】以‘嘎拉3’组培苗叶片愈伤组织为试材进行组培试验,设置培养基中NO3--N亏缺和适宜两个水平(NO3--浓度分别为0 mol/L和0.039 mol/L)。选取叶龄一致的功能性叶片,用灭菌手术刀片沿垂直叶脉方向划伤叶片并切除叶柄和叶尖,叶背向上平铺于MS分化培养基,暗培养3天然后转至光下7天,将长出愈伤组织的叶片分别转移至MS正常分化培养基(CK)和MS NO3--N亏缺分化培养基(T,用NH4Cl、KCl分别代替MS中的NH4NO3、KNO3),培养3周。在转板第0、1、3、7、14、21天分别取叶片伤口处的愈伤组织,观察其细胞形态,测定硝态氮含量、NO3-流速、氮素同化酶活性和氮素同化酶基因相对表达量。【结果】苹果愈伤组织经NO3--N亏缺处理1天后,细胞体积变小,间隙变大,排列疏松,7天后细胞变形,排列无规则。愈伤组织中硝态氮含量在处理7天时达到峰值,为1.54 mg/g,显著高于对照,最大降幅出现在7天后,为13.64%。NO3--N亏缺处理前,NO3-吸收速率最大,为22.38 pmol/(cm2·s),处理1天后降幅为84.1%,处理至7天时,NO3-已经由吸收变为外排,逆差为24.45 pmol/(cm2·s)。NR活性在处理至7天时无显著变化,7天后快速增加,增幅为19.26%。NiR活性在处理至14天时,无显著性差异,14天后上升幅度为21.83%,缺氮处理1天后,GS活性最低,为0.22U/g,7天后稍有增加,增幅为22.9%。处理组GOGAT活性在第3天时最低,为0.088 U/g,随后酶活性增加并保持稳定,但是仍低于对照组。处理组氮代谢关键酶基因MdNR2、MdNIR、MdGS2、MdGOGAT的表达量在处理至21天时达到峰值,分别为对照组表达量的3.36、2.52、11.37和2.29倍。【结论】苹果愈伤组织对缺氮非常敏感,从第一天起就�【Objectives】Investigation of the growth,nitrogen uptake and assimilation of apple callus under nitrogen deficiency can help us to understand the response mechanism of apple growth and development,and provide theoretical basis for further study of molecular mechanism of nitrogen deficiency affecting callus.【Methods】Leaf callus of‘Gala 3’apple induced from tissue culture seedlings were used as tested materials.Differentiation medium(MS)with deficient and normal levels of NO3--N(0 mol/L and 0.039 mol/L)were prepared,and the NH4NO3 and KNO3 in the deficient MS were replaced by NH4Cl and KCl.The same age of functional leaves were selected,and cut perpendicular to the vein and removed the petioles and tips using the sterilized surgical blade.The back of leaves were laid flat on MS normal differentiation medium,dark cultured for3 days and then 7 days under light.The leaves were transferred to prepared MS medium immediately and cultured for 3 weeks.The callus was collected from the wound of leaves after cultured for 0,1,3,7,14,21 days.The cell morphology,NO3--N content,NO3-uptake flux,N assimilation enzyme activity and relative expression of N assimilation enzyme genes were observed and tested.【Results】One day after NO3--N deficiency treatment,the cells of callus became smaller in volume,and arranged in loose with larger intercellular space.After 7 days,the cells were deformed and arranged irregularly.After 7 days,the NO3-content in callus reached the peak at 1.54 mg/g,which was significantly higher than that of the control.The maximum decrease rate was 13.64%after 7 days.Before NO3--N deficiency treatment,the NO3-absorption rate was the highest,which was 22.38 pmol/(cm2·s).With the prolongation of treatment time,the absorption rate decreased sharply and the decrease in the first treatment day was as high as 84.1%.After treatment for 7 days,NO3-was changed from uptake to efflux and the deficit was 24.45 pmol/(cm2·s).There was no significant change in NR activity within 7 days treatment,while increased r

关 键 词：苹果 氮亏缺 氮流量 氮同化 基因表达 

分 类 号：S51[农业科学—作物学]