本文選題：SR4　切入點：OMT4　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：土壤鹽堿化問題愈發(fā)嚴重,現(xiàn)在逐漸威脅到了農(nóng)作物的種植。土壤的鹽化與堿化總是相伴而生,與中性鹽給植物帶來的滲透脅迫和離子損傷相比,堿性鹽(Na2CO3和NaHCO3)還會導(dǎo)致土壤pH升高,破壞土壤結(jié)構(gòu),影響植物對離子的正常吸收造成細胞內(nèi)離子平衡穩(wěn)態(tài)被打破,破壞細胞膜的結(jié)構(gòu)和功能,最終會阻礙植物正常生長。到目前為止,人們更關(guān)注植物鹽脅迫響應(yīng)機制的研究,而有關(guān)植物耐堿分子機理的研究卻寥寥無幾,鑒于土壤的鹽漬化對植物造成的傷害比單一的鹽害更加嚴重,對植物堿脅迫的分子響應(yīng)機制的研究顯得尤為迫切。本實驗室前期利用不對稱體細胞雜交的技術(shù),獲得了一系列小麥/長穗偃麥草漸滲系新品系。山融4號(SR4)即是從中篩選的一個耐鹽堿小麥新品系。山融4號的耐堿能力已在田間實驗中得到了證實。我們從SR4堿處理后的轉(zhuǎn)錄組數(shù)據(jù)中發(fā)現(xiàn),氧甲基轉(zhuǎn)移酶TaOMT4和甘油三磷酸�；D(zhuǎn)移酶TaGPA6表達明顯上調(diào),因此,我們對其功能進行研究。1.小麥堿脅迫應(yīng)答基因TaOMT4的克隆與功能研究我們以SR4的cDNA為模板擴增得到TaOMT4基因,其開放閱讀框全長為1072 bp,編碼356個氨基酸。TaOMT4在堿脅迫下表達上調(diào)。亞細胞定位結(jié)果顯示,TaOMT4定位于細胞質(zhì)中。通過浸花法將TaOMT4轉(zhuǎn)入野生型擬南芥,篩選出兩個表達量較高的TaOMT4過表達純系進行耐逆相關(guān)研究。在高pH、堿性鹽(NaHCO3)和過氧化氫處理下,TaOMT4過表達系都表現(xiàn)出明顯的抗性。DAB染色結(jié)果顯示,TaOMT4過表達系的H2O2含量低于Col-0。TaOMT4過表達系的CAT和POD活性顯著高于對照株系。因此,我們推測TaOMT4可能使得抗氧化產(chǎn)物活性提升,導(dǎo)致植物體內(nèi)H2O2含量下降,從而提高植物對堿脅迫的耐受性。鹽處理的表型實驗結(jié)果發(fā)現(xiàn),TaOMT4過表達系對鹽脅迫的耐受性下降。qRT-PCR分析結(jié)果顯示,TaOMT4過表達系Na+/H+離子轉(zhuǎn)運體AtNHX1、AtNHX2表達量下降。按照以上結(jié)果我們推測TaOMT4可能通過負調(diào)控Na+轉(zhuǎn)運蛋白進而減弱植物的耐鹽性。TaOMT4過表達系對MV處理敏感。NBT染色結(jié)果顯示,TaOMT4過表達系的O2-水平明顯高于Col-0。TaOMT4過表達系的SOD酶活性無明顯變化。根據(jù)以上實驗結(jié)果,我們推測TaOMT4過表達系體內(nèi)O2-含量較高而SOD酶活不變可能與TaOMT4過表達系對MV敏感的表型相關(guān)。2.小麥堿脅迫應(yīng)答基因TaGPAT6的克隆與功能研究以SR4的cDNA為模板擴增得到TaGPAT6基因,其開放閱讀框全長為1494 bp,編碼497個氨基酸。TaGPAT6在堿脅迫下表達上調(diào)。亞細胞定位結(jié)果顯示,TaGPAT6定位于細胞質(zhì)中。通過浸花法將TaGPAT6轉(zhuǎn)入野生型擬南芥,篩選出兩個表達量較高的TaGPAT6過表達純系進行耐逆相關(guān)研究。在高pH、堿性鹽(NaHC03)和外源ABA處理下,TaGPAT6過表達系的根和葉片都表現(xiàn)出敏感的表型。同時,在高鹽處理下,TaGPAT過表達系表現(xiàn)出對高鹽的敏感性。ABA通路的相關(guān)Marker基因qRT-PCR分析結(jié)果顯示,過表達株系的AtCNED3、AtMYB2及AtMYC2的表達量顯著低于野生型,而ABA通路中正調(diào)控基因AtABI4、AtABI5的表達量明顯較高。離子轉(zhuǎn)運體相關(guān)Marker基因qRT-PCR分析結(jié)果顯示,在正常條件下過表達系A(chǔ)tHKT1;1表達量顯著低于Col-0。以上結(jié)果表明,TaGPAT6OE系對堿脅迫的響應(yīng)可能與ABA信號通路存在關(guān)系;TaGP4T6可能通過上調(diào)ABI4負調(diào)控HKT1;1從而在植物對鹽脅迫的響應(yīng)過程產(chǎn)生影響。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S512.1

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