本文關(guān)鍵詞：植物衰老控制基因克隆分析及葉片衰老遺傳調(diào)控　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 葉片衰老 擬南芥 LRR-RLKs基因 煙草 乙醇 NAP基因 誘導(dǎo)基因表達(dá)

【摘要】：衰老作為葉片發(fā)育的最后一個(gè)時(shí)期,其過程高度有序,同時(shí)又可被大量衰老相關(guān)基因(SAGs)嚴(yán)格調(diào)控。研究表明,衰老過程中會(huì)發(fā)生細(xì)胞程序性凋亡及營(yíng)養(yǎng)物質(zhì)的再分配,因此,葉片衰老對(duì)作物產(chǎn)量、品質(zhì)具有重要影響,因此,葉片衰老的研究具有重要理論意義及應(yīng)用價(jià)值。本研究以模式植物擬南芥和煙草為研究對(duì)象,初步探討了葉片衰老分子及遺傳調(diào)控機(jī)理,主要研究?jī)?nèi)容包括通過篩選LRR-RLK(富含亮氨酸類受體激酶)基因T-DNA插入突變體,明確了LRR-RLK基因在葉片衰老過程中的重要作用,同時(shí)對(duì)兩個(gè)LRR-RLK基因SRK1,SRK2進(jìn)行了功能驗(yàn)證,另外,本研究利用乙醇誘導(dǎo)型啟動(dòng)子誘導(dǎo)NAP基因表達(dá),初步探索生產(chǎn)上實(shí)現(xiàn)人工調(diào)控衰老的可行技術(shù)。主要結(jié)果包括：(1)篩選參與葉片衰老的擬南芥LRR-RLK基因,并對(duì)SRK1及SRK2進(jìn)行功能驗(yàn)證已有報(bào)道,類受體激酶在植物發(fā)育及抗性反應(yīng)中具有重要作用。LRR-RLK是其中一類最大的亞家族。然而,LRR-RLK是否參與了葉片衰老過程鮮有報(bào)道。本研究通過PCR方法鑒定到了26個(gè)LRR-RLK基因純合T-DNA插入突變體,進(jìn)一步通過衰老相關(guān)表型分析,發(fā)現(xiàn)其中6個(gè)LRR-RLK突變體能夠影響葉片衰老進(jìn)程,我們對(duì)其中2個(gè)基因SRK1(At4g08850),SRK2(At2g37050)進(jìn)行了較深入的功能驗(yàn)證。研究表明,SRKl及SRK2基因缺失突變體都表現(xiàn)為延遲衰老,葉綠素含量及FV/Fm比值等生理指標(biāo)進(jìn)一步驗(yàn)證了該結(jié)論；同時(shí),我們構(gòu)建了SRK1及SRK2的過量表達(dá)載體,并轉(zhuǎn)化野生型擬南芥;另外,我們分別提取野生型及srk1,srk2突變體的不同時(shí)期葉片(幼葉、完全伸展、衰老早起、衰老晚期)的RNA,反轉(zhuǎn)錄獲得cDNA,對(duì)SRK1,SRK2進(jìn)行表達(dá)模式分析。(2)在煙草中構(gòu)建誘導(dǎo)表達(dá)模式的葉片衰老可控體系誘導(dǎo)系統(tǒng)可使目的基因在合適的時(shí)空實(shí)現(xiàn)表達(dá)。轉(zhuǎn)錄因子NAP基因已被報(bào)道可在多種植物/作物中調(diào)控葉片衰老,因此,本研究探索了通過誘導(dǎo)表達(dá)NAP基因?qū)崿F(xiàn)衰老的人工調(diào)控。利用PCR方法分別從擬南芥和煙草中克隆到NAP基因的CDS序列,通過酶切連接的方式構(gòu)建了AlcA啟動(dòng)子驅(qū)動(dòng)的NAP基因的誘導(dǎo)表達(dá)載體；通過農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法獲得T0代轉(zhuǎn)基因煙草；對(duì)轉(zhuǎn)基因煙草噴灑03%乙醇,10天后發(fā)現(xiàn)與對(duì)照相比,轉(zhuǎn)基因植株有明顯的早衰表型；另外,對(duì)分離的葉片進(jìn)行相同處理,兩周后乙醇處理的材料較對(duì)照有明顯早衰表型；本研究結(jié)果為生產(chǎn)上實(shí)現(xiàn)人工可控的衰老模式提供了理論依據(jù)。本研究通過篩選擬南芥LRR-RLK突變體,明確了LRR-RLK基因在葉片衰老過程中具有重要作用,并對(duì)SRKl和SRK2進(jìn)行了功能驗(yàn)證；另外,我們構(gòu)建乙醇誘導(dǎo)NAP基因表達(dá)的轉(zhuǎn)基因煙草,并對(duì)轉(zhuǎn)基因植株進(jìn)行了乙醇誘導(dǎo)表型觀察,初步明確了生產(chǎn)上實(shí)現(xiàn)人工可控衰老調(diào)控模式的可行性。本文研究結(jié)果對(duì)深入理解葉片衰老調(diào)控機(jī)理具有重要意義,同時(shí)也為生產(chǎn)上應(yīng)用衰老可控技術(shù)實(shí)現(xiàn)作物高產(chǎn)提供一定的理論依據(jù)。
[Abstract]:As the last period of leaf development, senescence is highly orderly and can be strictly regulated by a large number of aging related genes (SAGs). Studies show that programmed cell apoptosis and redistribution of nutrients can occur during aging. Therefore, leaf senescence has an important impact on crop yield and quality. Therefore, leaf senescence research is of great theoretical significance and application value. In this study, Arabidopsis and tobacco as the research object, to explore the molecular and genetic mechanism of leaf senescence, the main research contents include the selection of LRR-RLK (leucine rich receptor kinase) gene T-DNA insertion mutants, clear LRR-RLK gene plays an important role in leaf senescence, while the two LRR-RLK gene SRK1. The SRK2 function has been verified, in addition, this study uses ethanol inducible promoter induced NAP gene expression and explore feasible implementation of artificial aging on production control. The main results are as follows: (1) screening Arabidopsis thaliana LRR-RLK genes involved in leaf senescence, and functional verification of SRK1 and SRK2 have been reported. Receptor like kinases play an important role in plant development and resistance response. LRR-RLK is one of the largest subfamilies. However, it is rarely reported whether LRR-RLK participates in the process of leaf senescence. This study through the PCR method identified 26 LRR-RLK gene homozygous T-DNA insertion mutants, through further analysis of aging related phenotypes, found that 6 LRR-RLK mutant can affect leaf senescence, we identified 2 genes SRK1 (At4g08850), SRK2 (At2g37050) for a more in-depth functional verification. Research shows that SRKl and SRK2 gene deletion mutants showed delayed senescence, chlorophyll content and the ratio of FV/Fm and other physiological indicators to further validate the conclusion; at the same time, we constructed the overexpression vector of SRK1 and SRK2, and transformed into wild type Arabidopsis; in addition, we do not extract wild type and srk1 mutant srk2 leaves in different periods (the young leaves, fully extended, early aging, aging late) RNA, reverse transcription cDNA, SRK1, SRK2 expression pattern analysis. (2) the induction system of leaf senescence controlled by the induction of expression pattern in tobacco can make the target gene express in the appropriate time and space. Transcription factor NAP gene has been reported to regulate leaf senescence in many plants / crops. Therefore, this study explored artificial control of senescence by inducing expression of NAP gene. By the method of PCR were cloned from Arabidopsis and tobacco to the CDS sequence of NAP gene, by enzyme connection way to construct the AlcA expression vector of NAP gene promoter induced promoter driven; generation of T0 transgenic tobacco by Agrobacterium mediated method; transgenic tobacco sprayed with 03% ethanol, and found 10 days later compared to the control, the transgenic plants have obvious senescence phenotype; in addition, the same treatment leaves on separation, two weeks after ethanol treatment materials have obvious premature aging phenotype compared with the control; the results of this study provides a theoretical basis for the production of artificial aging controlled mode. This study through the screening of the Arabidopsis LRR-RLK mutant, the LRR-RLK gene plays an important role in the senescence of leaves, and the SRKl and SRK2 to verify the function; in addition, we constructed transgenic tobacco ethanol induced expression of NAP gene, and the transgenic plants were observed phenotype induced by ethanol, initially identified the production feasibility of artificial control aging regulation mode. The results of this study are important for understanding the mechanism of leaf senescence control, and also provide a theoretical basis for the application of aging control technology to achieve high yield.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2
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本文編號(hào)：1347285