【作者】 刘晓；

【导师】 张数鑫；

【作者基本信息】 山东农业大学 ， 生物学， 2022， 硕士

【摘要】 玉米作为我国第一大粮食作物,产量仅次于美国,位居世界第二。低温、盐碱、高温等非生物胁迫严重影响玉米的产量和品质。目前我国盐碱地面积约为15亿亩,其中可利用的盐碱地面积约为5.5亿亩,有2亿亩具备农业开发的潜力,提高玉米抗盐性,能够提高土地利用率,实现盐碱地的高效利用。此外,低温一方面限制玉米在高纬度栽培,另一方面对生产上种子的萌发造成严重影响,提高玉米抗冻性有助于提前播种获得高质量大田群体,并有可能使春播玉米种植区域北移,对指导我国玉米生产有重要意义。植物在低温胁迫和盐胁迫环境下,会触发自身应激响应机制,通过不同途径帮助植物抵御逆境胁迫。ABC转运蛋白常见于原核生物和真核生物的一类跨膜转运蛋白,广泛参与植物逆境胁迫响应过程,本课题对玉米ABC转运蛋白ZmMRPA6基因抵抗低温胁迫和盐胁迫的响应机制进行了初步探究,主要研究结果如下:(1)通过玉米种子耐冷萌发的全基因组关联分析筛选到一个编码ABC转运蛋白的耐冷候选基因ZmMRPA6,结合之前报道的玉米盐胁迫转录组分析数据,发现ZmMRPA6在盐胁迫下转录水平表达下调,随后对ZmMRPA6在玉米抵抗低温和盐胁迫过程中发挥的作用进行实验探索。(2)通过搜索EMS诱变突变体库订购到zmmrpa6突变体,该突变体的第6内含子的剪接识别位点发生了一个C到T单碱基突变,导致突变体内大部分转录本的该内含子无法正常剪切,基因和蛋白结构发生变化,蛋白翻译因第6内含子内的终止密码子提前终止,导致蛋白功能丧失。(3)系统分析了低温胁迫和盐胁迫下zmmrpa6突变体萌发期和幼苗期的表型,结合萌发率和根长的数据统计,发现zmmrpa6突变体萌发期表现出显著的抗冻性和抗盐性;结合叶片离子渗漏分析、相对损伤面积和活性氧含量,验证了zmmrpa6突变体在苗期也具有抗冻性和抗盐性。(4)qRT-PCR检测zmmrpa6突变体在盐和低温胁迫下ZmMRPA6基因和部分响应胁迫的功能基因转录水平表达,发现zmmrpa6突变体与B73中转录水平表达模式一致,zmmrpa6内含子滞留突变体不影响转录因子ZmmICE1基因、硬脂酰载体蛋白脱氢酶ZmSAD基因、过氧化氢酶ZmCAT基因、抗坏血酸氧化酶ZmAPX基因、超氧化物歧化酶ZmSOD基因和促分裂原活化蛋白磷酸激酶ZmMPK5基因的表达。(5)通过蛋白质谱分析发现突变体zmmrpa6与B73中的差异蛋白大部分参与氧化还原、水解酶活性、酸类以及小分子代谢和质膜代谢等参与胁迫响应的代谢途径,这也为zmmrpa6突变体调控玉米抗冷和抗盐提供了蛋白水平的依据。(6)通过瞬时表达烟草叶片细胞验证了ZmMRPA6基因编码的ABC转运蛋白ZmMRPA6定位在细胞膜。这些结果表明zmmrpa6突变体不是通过转录水平提高玉米抗冷和抗盐能力,可能通过蛋白物质跨膜转运能力的基本丧失来增强玉米抗盐和抗冻能力。(7)对拟南芥AtABCC4和AtABCC14基因(ZmMRPA6基因的同源基因)T-DNA插入突变体的萌发期和莲座叶期幼苗进行了抗盐性评价,发现拟南芥突变体对盐胁迫敏感,同时AtABCC4和AtABCC14亚细胞定位与ZmMRPA6蛋白一致,均定位在细胞膜上。综上所述,ZmMRPA6基因在玉米抗冻和抗盐调控信号通路中发挥着重要作用,其第6内含子滞留会导致蛋白功能丧失进而提高玉米抗冻和抗盐能力。需要结合ZmMRPA6基因过表达株系进一步分析探究ZmMRPA6基因的功能。更多还原

【Abstract】 Maize,the largest food crop in China,is the second largest in the world after the United States.Low temperature,salinity,high temperature and other abiotic stresses seriously affect the yield and quality of maize.Atpresent,the area of saline-alkali land in China is about 1.5billion mu,of which the available area is about 550 million mu,and 200 million mu have the potential of agricultural development.Improving the salt resistance of maize can improve the land utilization rate and realize the efficient utilization of saline-alkali land.In addition,on the one hand,low temperature limits maize cultivation at high latitudes,on the other hand,it has a serious impact on seed germination in production.Improving the frost resistance of maize is helpful for early sowing to obtain high quality field populations,and it is possible to move the planting area of spring maize northward,which is of great significance to guide maize production in China.Under low temperature stress and salt stress,plants trigger their own stress response mechanism and help plants resist adversity stress through different ways.ABC transporters are common in prokaryotes and eukaryotes,and are widely involved in plant stress response.In this study,the response mechanism of maize ABC transporter ZmMRPA6 to low temperature stress and salt stress was preliminarily explored.The main results are as follows:(1)A cold-tolerant candidate gene ZmMRPA6 encoding ABC transporter was screened by genome-wide association analysis of maize seed germination under cold stress.Combined with the previously reported transcriptome analysis data of maize under salt stress,it was found that the expression of ZmMRPA6 was down-regulated at the transcriptional level under salt stress.Subsequently,the role of ZmMRPA6 in maize resistance to low temperature and salt stress was experimentally explored.(2)The zmmrpa6 mutant was ordered by searching the EMS mutagenesis mutant library.A single base mutation from C to T occurred in the splicing recognition site of the sixth intron of the mutant,resulting in the intron of most transcripts in the mutant could not be cut normally,and the gene and protein structure changed.Protein translation was terminated in advance due to the termination codon in the sixth intron,resulting in the loss of protein function.(3)Phenotypes at germination and seedling stages of zmmrpa6 mutant under low temperature stress and salt stress were systematically analyzed.Combined with the statistics of germination rate and root length,it was found that zmmrpa6 mutant showed obvious frost resistance and salt resistance at germination stage.Combined with leaf ion leakage analysis,relative damage area and active oxygen content,it was verified that zmmrpa6 mutant also had frost resistance and salt resistance at seedling stage.(4)The transcription levels of ZmMRPA6 gene and some functional genes responding to salt and low temperature stress in zmmrpa6 mutant were detected by qRT-PCR.It was found that the transcription level expression pattern of zmmrpa6 mutant was consistent with that in B73.The intron retention mutant of zmmrpa6 did not affect the expression of transcription factor ZmmICE1 gene,stearoyl carrier protein dehydrogenase ZmSAD gene,catalase ZmCAT gene,ascorbic acid oxidase ZmAPX gene,superoxide dismutase ZmSOD gene and mitogen-activated protein kinase ZmMPK5 gene.(5)Protein spectrum analysis showed that most of the differentially expressed proteins in zmmrpa6 and B73 were involved in redox,hydrolytic enzyme activity,acid metabolism,small molecule metabolism and plasma membrane metabolism,which also provided a protein level basis for zmmrpa6 mutant to regulate maize cold and salt resistance.(6)The transient expression of tobacco leaf cells verified that the ABC transporter ZmMRPA6 encoded by ZmMRPA6 gene was localized to the cell membrane.These results indicated that the zmmrpa6 mutant did not improve the cold and salt resistance of maize at the transcriptional level,and it might enhance the salt and frost resistance of maize through the basic loss of protein transmembrane transport capacity.(7)The T-DNA insertion mutants of AtABCC4 and AtABCC14 genes(homologous genes of ZmMRPA6 gene)were evaluated for salt tolerance at germination stage and rosette leaf stage.It was found that Arabidopsis mutants were sensitive to salt stress.AtABCC4 and AtABCC14 subcellular localization and ZmMRPA6 protein were located on the cell membrane.In summary,ZmMRPA6 gene plays an important role in the regulation signaling pathway of maize frost resistance and salt resistance.The retention of its sixth intron leads to the loss of protein function and thus improves the frost resistance and salt resistance of maize.The function of ZmMRPA6 gene needs to be further analyzed in combination with the overexpression lines of ZmMRPA6.更多还原