【作者】 张玉华；

【导师】 陈凡国；

【作者基本信息】 山东大学 ， 生物工程（专业学位）， 2018， 硕士

【摘要】 高盐度和干旱可以诱导植物体产生过量的活性氧(ROS),导致对DNA、蛋白质和脂类等生物大分子的氧化损伤。而蛋白质中的含硫氨基酸甲硫氨酸(Met)很容易被ROS氧化,形成S型甲硫氨酸亚砜(Met-S-SO)和R型甲硫氨酸亚砜(Met-R-SO)。机体内的甲硫氨酸亚砜还原酶(methionine sulfoxide reductase,MSR)可以还原氧化型的甲硫氨酸。虽然已有研究表明生物中的MSR可以响应多种逆境反应,但对于玉米中的MSR的作用缺少实验证据,本论文主要对ZmMSRA2和-A5.1的抗逆功能及作用机制进行了初步探究。1 ZmSRA2的功能及作用机制生物信息学分析显示,ZmMSRA2编码蛋白包含一个高度保守的C端的PMSR结构域,亚细胞定位结果显示ZmMSRA2定位在细胞质,酶活测定结果显示过表达系的MSR酶活与野生型相比显著提高。前期qPCR分析发现NaCl、PEG处理后,ZmMSRA2表达上调;在NaCl、Mannitol、H202以及ABA处理下,转基因拟南芥株系的主根长度显著增加,耐逆能力强于对照。在渗透胁迫处理下,与野生型相比,过表达系种子萌发较快。测量各株系的抗氧化酶活性和脯氨酸的含量,结果显示,过表达ZmMSRA2基因可显著提高转基因拟南芥中的CAT酶活及脯氨酸的含量。通过对相关代谢过程marker基因分析,结果表明ZmMSRA2促进了ROS清除相关基因、ABA合成相关基因和吡咯啉-5-羧酸合成酶基因(P5CS1)的表达。以上结果显示,玉米ZmMSRA2通过调控脯氨酸合成及ROS的含量响应逆境的胁迫。2 ZmMRA5.1的功能及作用机制生物信息学分析显示,ZmMSRA5.1编码的蛋白主要由N端的低复杂性区域(low complexity region)和C端的PMSR结构域组成,亚细胞定位结果显示ZmMSRA5.1可能分泌到胞外,酶活测定结果表明过表达系的MSR酶活比对照显著提高。前期qPCR分析发现NaCl、PEG处理后,ZmMSRA5.1表达上调,在NaCl、Mannitol、H2O2处理下,与对照相比,转基因株系的抗胁迫能力显著提高,盐胁迫处理下过表达系种子的萌发率以及萌发速度较野生型都有一定程度的提高。测量各株系的氧化酶活和脯氨酸的含量,结果显示,过表达ZmMSRA5.1基因可显著提高转基因拟南芥中的CAT酶活。通过对ROS代谢过程marker基因分析,结果表明ZmMSRA5.1提高了ROS清除系统关键酶的表达;ZmMSRA5.1过表达系的离子通道蛋白相关marker基因SOS3、NHX1表达量显著高于对照。因此,ZmMSRA5.1可能通过正调控离子转运载体及ROS含量响应逆境胁迫。更多还原

【Abstract】 High salinity and drought can induce excess reactive oxygen species(ROS),which could cause oxidative damage to DNA,proteins,lipids and other biological macromolecules.The sulfur-containing amino acid in protein,such as Met,is very easily oxidized by ROS to form S-methionine sulfoxide(Met-S-SO)and R-methionine sulfoxide(Met-R-SO).The methionine sulfoxide reductases(MSRs)could reduce the oxidative methionine to Met.Although MSR involved in multiple stress responses,there is no experimental evidence for the role of MSR in maize.This paper is mainly to discover the functions and their mechanisms of ZmMSRA2 and-A5.1.1 The function and mechanism of ZmMSRA2Bioinformatics analysis showed that the predicted protein ZmMSRA2 contains a highly conserved PMSR domain in C-terminus.The subcellular localization results showed that ZmMSRA2 was located in the cytosol.The results of enzyme activity assay showed that the MSR activity of the overexpressed lines were significantly higher than that of the wild one.qPCR analysis in previous study showed that the expression of ZmMSRA2 was up-regulated after NaCl and PEG treatment.Under the treatment of NaCl,Mannitol,H2O2,and ABA,the length of the main root in overexpressors was significantly increased,and the stress tolerance of the transgenic lines was stronger than that of the control.Under the osmotic stress treatment,the speed of germination in the overexpressiors were increased compared to those of the wild one.The CAT activity and proline content were measured.It was shown that CAT enzyme activity and proline content increased significantly in A.thaliana plants which constitutively expressing ZmMSRA2 than those of control.By analyzing the relevant marker genes,it was found that the expressions of ROS scavenging genes,ABA synthesis related genes,and pyrroline-5-carboxylic acid synthase gene(P5CS1)increased obviously than those in control.The above results show that maize ZmMSRA2 involved in stress tolerance via modulating proline synthesis and ROS content.2 The function and mechanism of ZmMSRA5.1Bioinformatic analysis showed that the predicted protein ZmMSRA5.1 is composed of a low complexity region at the N-terminus and a PMSR domain at the C-terminus.Subcellular localization results showed that ZmMSRA5.1 may be a secreted protein.The enzyme activity assay shown that the MSR enzyme activity in overexpressor was significantly increased compared to the control.qPCR analysis in previous work showed that the expression of ZmMSRA5.1 was up-regulated after NaCl or PEG treatment.Under NaCl,Mannitol and H2O2 treatment,the stress tolerance of the transgenic lines was significantly stronger than that of the control,and the germination rate and speed of the over-expressed seeds under salt stress were higher than that of the wild one.The CAT activity and proline content were measured.The results showed that overexpression of ZmMSRA5.1 gene in Arabidopsis promoted the CAT activity significantly in transgenic lines than in control.Through the analysis of the expression of marker genes,it was shown that the expression of ROS scavenging genes and ion channel protein related marker gene SOS3,NHX1 increased signific-antly in transgenic lines than in control.Therefore,ZmMSRA5.1 responded to abiotic stress by positively regulating ion transporter and ROS content.更多还原