小麦碱胁迫应答基因TaOMT4和TaGPAT6的功能研究

发布时间：2018-03-14 01:13

本文选题：SR4　切入点：OMT4　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：土壤盐碱化问题愈发严重,现在逐渐威胁到了农作物的种植。土壤的盐化与碱化总是相伴而生,与中性盐给植物带来的渗透胁迫和离子损伤相比,碱性盐(Na2CO3和NaHCO3)还会导致土壤pH升高,破坏土壤结构,影响植物对离子的正常吸收造成细胞内离子平衡稳态被打破,破坏细胞膜的结构和功能,最终会阻碍植物正常生长。到目前为止,人们更关注植物盐胁迫响应机制的研究,而有关植物耐碱分子机理的研究却寥寥无几,鉴于土壤的盐渍化对植物造成的伤害比单一的盐害更加严重,对植物碱胁迫的分子响应机制的研究显得尤为迫切。本实验室前期利用不对称体细胞杂交的技术,获得了一系列小麦/长穗偃麦草渐渗系新品系。山融4号(SR4)即是从中筛选的一个耐盐碱小麦新品系。山融4号的耐碱能力已在田间实验中得到了证实。我们从SR4碱处理后的转录组数据中发现,氧甲基转移酶TaOMT4和甘油三磷酸酰基转移酶TaGPA6表达明显上调,因此,我们对其功能进行研究。1.小麦碱胁迫应答基因TaOMT4的克隆与功能研究我们以SR4的cDNA为模板扩增得到TaOMT4基因,其开放阅读框全长为1072 bp,编码356个氨基酸。TaOMT4在碱胁迫下表达上调。亚细胞定位结果显示,TaOMT4定位于细胞质中。通过浸花法将TaOMT4转入野生型拟南芥,筛选出两个表达量较高的TaOMT4过表达纯系进行耐逆相关研究。在高pH、碱性盐(NaHCO3)和过氧化氢处理下,TaOMT4过表达系都表现出明显的抗性。DAB染色结果显示,TaOMT4过表达系的H2O2含量低于Col-0。TaOMT4过表达系的CAT和POD活性显著高于对照株系。因此,我们推测TaOMT4可能使得抗氧化产物活性提升,导致植物体内H2O2含量下降,从而提高植物对碱胁迫的耐受性。盐处理的表型实验结果发现,TaOMT4过表达系对盐胁迫的耐受性下降。qRT-PCR分析结果显示,TaOMT4过表达系Na+/H+离子转运体AtNHX1、AtNHX2表达量下降。按照以上结果我们推测TaOMT4可能通过负调控Na+转运蛋白进而减弱植物的耐盐性。TaOMT4过表达系对MV处理敏感。NBT染色结果显示,TaOMT4过表达系的O2-水平明显高于Col-0。TaOMT4过表达系的SOD酶活性无明显变化。根据以上实验结果,我们推测TaOMT4过表达系体内O2-含量较高而SOD酶活不变可能与TaOMT4过表达系对MV敏感的表型相关。2.小麦碱胁迫应答基因TaGPAT6的克隆与功能研究以SR4的cDNA为模板扩增得到TaGPAT6基因,其开放阅读框全长为1494 bp,编码497个氨基酸。TaGPAT6在碱胁迫下表达上调。亚细胞定位结果显示,TaGPAT6定位于细胞质中。通过浸花法将TaGPAT6转入野生型拟南芥,筛选出两个表达量较高的TaGPAT6过表达纯系进行耐逆相关研究。在高pH、碱性盐(NaHC03)和外源ABA处理下,TaGPAT6过表达系的根和叶片都表现出敏感的表型。同时,在高盐处理下,TaGPAT过表达系表现出对高盐的敏感性。ABA通路的相关Marker基因qRT-PCR分析结果显示,过表达株系的AtCNED3、AtMYB2及AtMYC2的表达量显著低于野生型,而ABA通路中正调控基因AtABI4、AtABI5的表达量明显较高。离子转运体相关Marker基因qRT-PCR分析结果显示,在正常条件下过表达系AtHKT1;1表达量显著低于Col-0。以上结果表明,TaGPAT6OE系对碱胁迫的响应可能与ABA信号通路存在关系;TaGP4T6可能通过上调ABI4负调控HKT1;1从而在植物对盐胁迫的响应过程产生影响。
[Abstract]:The problem of soil salinization has become more serious, now gradually threaten crops. Soil salinization and alkalization is always accompanied with infiltration and salt to bring the plant stress and ion injury compared with alkaline salts (Na2CO3 and NaHCO3) can lead to increased soil pH, destroying the soil structure, affecting the normal plants on ion absorption ion homeostasis in cells caused by balance is broken, the structure and function of cell membrane damage, will eventually hinder the normal growth of plants. So far, people pay more attention to research on plant salt stress response mechanism, and study the molecular mechanism of plant resistance is scanty, because of Soil Salinization on plant damage than single salt more seriously, the alkaloid molecular stress response mechanism research is particularly urgent. Ourprevious using asymmetric somatic hybridization technique, obtained a series of The column of wheat / e.elongata introgression lines. Shanrong No. 4 (SR4) is a salt tolerant wheat varieties selected from. Shanrong No. 4 alkali ability has been in the field experiment. We found from the transcriptome data of SR4 after alkali treatment, oxygen methyltransferase TaOMT4 and glycerol three phosphate acyltransferase TaGPA6 expression was up-regulated, therefore, we studied the cloning and functional study of.1. wheat alkali stress responsive gene TaOMT4 using SR4 cDNA as template to amplify TaOMT4 gene on its function, its open reading frame was 1072 BP, encoding 356 amino acids of.TaOMT4 in alkali stress under expression. Subcellular localization showed that TaOMT4 localized in the cytoplasm. The flower dipping method TaOMT4 into wild type Arabidopsis, selected two high expression of TaOMT4 expression clones related research tolerance. At high pH, alkali Salt (NaHCO3) and hydrogen peroxide treatment, TaOMT4 overexpression showed obvious resistance to.DAB staining showed that the TaOMT4 content of H2O2 expression system was lower than that of CAT and the activity of POD Col-0.TaOMT4 overexpression was significantly higher than that of control plants. Therefore, we speculate that TaOMT4 may enhance the antioxidant activity of product making, leading to decreased H2O2 content in vivo plants to improve plant tolerance to alkali stress. The experimental results of phenotypic salt treatment showed that overexpression of TaOMT4 on salt tolerance by.QRT-PCR analysis showed that TaOMT4 overexpression Na+/H+ ion transporter AtNHX1, AtNHX2 expression decreased. According to the above results we speculate that TaOMT4 may through the negative regulation of Na+ transporter then.TaOMT4 decreased salt tolerance in plants by overexpression of MV sensitive.NBT staining showed that the expression of TaOMT4, O2- levels were significantly higher than that of Col-0.TaOMT4. The expression of SOD enzyme activity had no obvious change. According to the above results, we speculate that TaOMT4 had higher levels of O2- expression, SOD activity and TaOMT4 overexpression may change responsive gene TaGPAT6 on MV sensitive phenotype correlation.2. wheat alkali stress cloning and functional study of SR4 cDNA were amplified by TaGPAT6 gene and the open reading frame was 1494 BP, encoding 497 amino acids of.TaGPAT6 expression under alkali stress. Subcellular localization showed that TaGPAT6 located in the cytoplasm. The flower dipping method TaGPAT6 into wild type Arabidopsis, two screened the high expression of TaGPAT6 overexpression in pure research related tolerance in. High pH, alkaline salt (NaHC03) and exogenous ABA treatment, TaGPAT6 expression of root and leaf showed sensitive phenotype. At the same time, in the high salt treatment, TaGPAT overexpression showed high salt The sensitivity of Marker.ABA pathway related gene qRT-PCR analysis showed that overexpression strains of AtCNED3, expression of AtMYB2 and AtMYC2 was significantly lower than that of the wild type, while the ABA pathway is the regulation of gene AtABI4, AtABI5 expression was significantly higher. Transporter Marker gene qRT-PCR analysis showed that the overexpression of AtHKT1 in normal conditions 1; the expression was significantly lower than the above Col-0. results show that TaGPAT6OE system has relation to alkali stress response may be related to ABA signaling pathway; TaGP4T6 may regulate ABI4 negative regulation of HKT1; 1 and in plant responses to salt stress influence.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S512.1

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