富天冬酰胺蛋白增强拟南芥辐射抗性的研究

Ability of asparagine-rich protein to enhance the radiation resistance of Arabidopsis thaliana

目的研究富天冬酰胺蛋白(NRP)对拟南芥辐射抗性的影响。方法选取野生型(WT)、NRP过表达型(Pro35S:NRP-GFP)、NRP突变型(nrp)3种拟南芥种子,分别分为照射组(120 Gyγ射线照射)和对照组(不照射)。(1)3种基因型拟南芥种子培养至第3天进行照射,照射后继续培养至第7天,统计各组根长。(2)3种基因型拟南芥种子于MS培养基中培养至第7天进行照射,照射后分别移栽至基质土中继续培养至第30天,观察各组植株的生长和形态。(3)WT拟南芥种子培养至第7天进行照射,照射后继续培养24 h,采用实时定量PCR分析WT植株中NRP和多聚二磷酸腺苷核糖聚合酶2(PARP2)2种基因在照射前后的相对表达量的变化。(4)Pro35S:NRP-GFP拟南芥种子培养至第7天进行照射,分别于照射后不同时间取幼苗进行荧光共聚焦显微观察。组间比较采用独立样本t检验。结果(1)辐射导致WT和nrp幼苗的根长缩短,分别为(2.73±0.43)cm和(1.31±0.53)cm,与对照组[(4.56±0.41)cm和(2.89±0.60)cm]相比,差异均有统计学意义(t=9.212、6.490,均P=0.000);而Pro35S:NRP-GFP幼苗的根长在照射前后无明显差异[(3.01±0.34)cm vs.(2.96±0.34)cm,t=0.253,P=0.801]。(2)辐射导致WT和nrp幼苗植株的发育不良、生长受限,其中株高和莲座叶直径分别与对照组相比,差异均有统计学意义(t=6.361~12.250,均P=0.000),而Pro35S:NRP-GFP幼苗植株则维持正常形态。(3)辐射导致WT植株中NRP和PARP2的相对表达量均升高,与对照组相比,差异有统计学意义(t=4.447、7.776,P=0.002、0.000)。(4)Pro35S:NRP-GFP幼苗在照射后各时间点均出现NRP入核现象。结论NRP可能对拟南芥辐射抗性的发挥起到重要作用,可作为植物辐射抗性靶点进行深入研究。

Objective To investigate the role of asparagine-rich protein(NRP)in resistance of Arabidopsis thaliana to radiation.Methods Wild-type(WT),NRP overexpression lines(Pro35S:NRP-GFP),and NRP mutant lines(nrp)of A.thaliana seeds were used and divided into two groups:irradiation(120 Gyγ-ray irradiation)and control(no irradiation).(1)Seeds of three genotypes of A.thaliana were cultured to the third day and irradiated with 120 Gy;the root length of each group was measured after culture was continued to the seventh day.(2)The seeds of three genotypes of A.thaliana were cultured in MS medium to the seventh day,and then irradiated with 120 Gy.After irradiation,the seeds were transplanted to the substrate soil for further culture until the 30th day.The growth and morphology of each group of plants were observed.(3)WT A.thaliana seeds were cultured to the seventh day and irradiated with 120 Gy.After 24 h,the changes in the relative expression of NRP and poly-ADP-ribose polymerase 2(PARP2)genes in WT plants before and after irradiation were analyzed by real-time quantitative PCR.(4)The seeds of Pro35S:NRP-GFP A.thaliana were cultured to the seventh day and then irradiated with 120 Gy.The seedlings were observed by fluorescence confocal microscopy at different times after irradiation.Independent sample t-test was used for comparison between groups.Results(1)After irradiation,the root lengths of WT and nrp A.thaliana were significantly shortened(t=9.212 and 6.490,both P=0.000)to(2.73±0.43)cm and(1.31±0.53)cm,respectively,whereas the root lengths of the control group were(4.56±0.41)cm and(2.89±0.60)cm.The root lengths of Pro35S:NRP-GFP with or without irradiation exhibited no significant changes((3.01±0.34)cm vs.(2.96±0.34)cm,t=0.253,P=0.801).(2)The growth and development of WT and nrp A.thaliana were significantly reduced(t=6.361–12.250,all P=0.000),whereas that of Pro35S:NRP-GFP maintained its development after irradiation.(3)The relative expression of NRP and PARP2 of WT A.thaliana significantly increased after irradiation(t=4.447 and 7.776,P=0.002 and 0.000).(4)Pro35S:NRP-GFP seedlings exhibited NRP re-localization into the nucleus after irradiation at each time point.Conclusion NRP may play a critical role in the resistance of A.thaliana to radiation,suggesting that NRP may be an important gene in radiation-caused injury in this species.