本文選題：PLATZ5 + SOS3　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：土壤鹽漬化是影響當(dāng)今農(nóng)作物產(chǎn)量和質(zhì)量的一個(gè)重要因素。土壤鹽分濃度過(guò)高會(huì)導(dǎo)致植物代謝紊亂,影響植物對(duì)必需元素的吸收,造成滲透脅迫及氧化脅迫等次生脅迫,進(jìn)而影響植物正常的生長(zhǎng)發(fā)育。如何提高植物的耐鹽性已成為農(nóng)業(yè)發(fā)展急需解決的問(wèn)題。PLATZ(Plant A/T rich sequence-and zinc-binding protein)是一類植物特有的轉(zhuǎn)錄因子,2002年豌豆PLATZ1首次被報(bào)道能非特異地結(jié)合富含A/T堿基的序列并發(fā)揮轉(zhuǎn)錄抑制的作用。擬南芥中共有12個(gè)PLATZ成員,本研究對(duì)擬南芥AtPLATZ5進(jìn)行了表達(dá)分析和功能鑒定,結(jié)果如下:(1)利用半定量RT-PCR和GUS染色的方法檢測(cè)了AtPLATZ5的組織表達(dá)模式,結(jié)果表明AtPLATZ5在各個(gè)組織中均有表達(dá),且表達(dá)差異不大。RT-PCR和qRT-PCR的結(jié)果同時(shí)證明了AtPLATZ5的表達(dá)量受鹽脅迫誘導(dǎo)。(2)利用瞬時(shí)轉(zhuǎn)化煙草的方法對(duì)PLATZ5的亞細(xì)胞定位進(jìn)行了分析,結(jié)果表明PLATZ5除在細(xì)胞核中表達(dá)外,在細(xì)胞質(zhì)中也有表達(dá),這說(shuō)明PLATZ5可能發(fā)揮多種功能。(3)利用雙熒光素酶報(bào)告系統(tǒng)對(duì)PLATZ5的轉(zhuǎn)錄活性進(jìn)行了分析,結(jié)果表明PLATZ5具有轉(zhuǎn)錄抑制活性。(4)對(duì)AtPLATZ5超表達(dá)株系進(jìn)行100mM和150mM NaCl處理,發(fā)現(xiàn)AtPLATZ5超表達(dá)株系地上部鮮重比對(duì)照分別降低了30%與50%,而根部的鮮重比對(duì)照分別降低了20%與18%,這表明鹽脅迫對(duì)AtPLATZ5超表達(dá)株系地上部抑制更明顯。提高NaCl濃度到200mM時(shí),超表達(dá)株系葉片白化,存活率顯著低于野生型。這說(shuō)明AtPLATZ5負(fù)調(diào)控幼苗對(duì)鹽脅迫的抗性。AtPLATZ5超表達(dá)株系地上部也會(huì)對(duì)100mM NaNO3、100mM KCl和100mM KNO3脅迫處理表現(xiàn)出微弱敏感的表型,同時(shí)超表達(dá)株系對(duì)10mM LiCl超敏感而對(duì)甘露醇不敏感。以上結(jié)果表明AtPLATZ5主要響應(yīng)離子脅迫。(5)插入到外顯子區(qū)的platz5突變體對(duì)鹽脅迫處理沒(méi)有表型,表明PLATZ家族成員可能存在著功能冗余。(6)鈉離子探針染色結(jié)果顯示超表達(dá)株系在受到鹽脅迫時(shí)根與葉片部積累的鈉離子增多,表明超表達(dá)株系體內(nèi)鈉離子的吸收轉(zhuǎn)運(yùn)受到了影響。(7)qRT-PCR分析鹽脅迫后超表達(dá)株系體內(nèi)信號(hào)通路marker基因的變化,發(fā)現(xiàn)SOS途徑的SOS3/CBL4及其同源基因CBL10表達(dá)量下調(diào),而染色質(zhì)免疫共沉淀實(shí)驗(yàn)證明PLATZ5能靶向SOS3啟動(dòng)子,這表明PLATZ5通過(guò)抑制SOS3的表達(dá)來(lái)響應(yīng)鹽脅迫。
[Abstract]:Soil salinization is an important factor affecting crop yield and quality. The high concentration of soil salt will lead to the disorder of plant metabolism, which will affect the absorption of essential elements, osmotic stress, oxidative stress and other secondary stresses, and then affect the normal growth and development of plants. How to improve plant salt tolerance has become an urgent problem in agricultural development. The plant A / T rich sequence-and zinc-binding protein is a kind of plant-specific transcription factor. In 2002, pea PLATZ1 was first reported to be able to combine the sequence with rich in A / T base and play an important role. Transcriptional inhibition. There are 12 PLATZ members in Arabidopsis thaliana. In this study, the expression and function of Arabidopsis AtPLATZ5 were analyzed. The results are as follows: 1) the expression pattern of AtPLATZ5 was detected by semi-quantitative RT-PCR and GUS staining. The results showed that AtPLATZ5 was expressed in all tissues, and the difference was not significant. The results of RT-PCR and qRT-PCR also proved that the expression of AtPLATZ5 was induced by salt stress. (2) the subcellular localization of PLATZ5 was analyzed by transient transformation of tobacco. The results showed that PLATZ5 was expressed not only in the nucleus, but also in the cytoplasm, which suggested that PLATZ5 might play a variety of functions.) the transcriptional activity of PLATZ5 was analyzed by using double luciferase report system. The results showed that PLATZ5 had transcriptional inhibitory activity. 4) 100mM and 150mM NaCl were used to treat AtPLATZ5 overexpression lines. The results showed that the fresh weight of AtPLATZ5 superexpression lines was 30% and 50% lower than that of the control, while the fresh weight of roots was 20% and 18% lower than that of the control, respectively, which indicated that salt stress had more obvious inhibition on the shoot of AtPLATZ5 overexpressed lines. When the concentration of NaCl was increased to 200mM, the overexpressed lines were albino, and the survival rate was significantly lower than that of wild type. This indicated that the shoots of the seedlings with negative AtPLATZ5 regulation showed weak phenotypic sensitivity to 100mM NaNO3100mM KCl and 100mM KNO3 stress, but the overexpressed lines were not sensitive to mannitol but hypersensitive to 10mM LiCl. The results indicated that AtPLATZ5 was mainly responsive to ion stress. The platz5 mutant inserted into the exon region had no phenotype under salt stress. The results indicated that the PLATZ family members might have functional redundancy. 6) the results of sodium probe staining showed that the accumulation of sodium ions in roots and leaves of the overexpression lines increased under salt stress. The results showed that the absorption and transport of sodium ions in the overexpression lines were affected by QRT-PCR. The changes of marker gene in the signaling pathway of the overexpression lines after salt stress were analyzed. It was found that the expression of SOS3/CBL4 and its homologous gene CBL10 in the SOS pathway was down-regulated. The chromatin immunoprecipitation assay showed that PLATZ5 could target the SOS3 promoter, which suggested that PLATZ5 responded to salt stress by inhibiting the expression of SOS3.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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