發(fā)布時間：2018-04-08 17:47

  本文選題：玉米　切入點：蟲害　出處：《中國農業(yè)大學》2015年博士論文

【摘要】：玉米(Zea mays L.)作為重要的糧食作物和工業(yè)原料在國民經濟中占據(jù)重要地位。蟲害是制約玉米產量的關鍵因素之一。已有的研究結果表明玉米萜類合成酶在玉米的間接防御中起到非常重要的作用。玉米受到鱗翅目幼蟲為害后,葉片會釋放一系列的萜類化合物吸引害蟲的天敵。雖然在過去的二十年中玉米已經成為研究植物間接防御的一個模式系統(tǒng),但是對于萜類合成酶基因的表達調控機制在玉米中還未見報道。本研究以玉米萜類合成酶基因TPS10啟動子為主要研究對象,目的是闡明玉米萜類合成酶基因TPS10的表達調控機制。qPCR結果表明TPS10基因受蟲害和MeJA誘導表達,表達部位在葉片并且其表達水平在一定程度上受光合作用的影響。PLACE預測結果顯示1727 bp的TPS10啟動子序列中存在多個與植物防御相關的順式作用元件,如GCC-box,G-box,W-box。轉基因擬南芥的GUS組織化學染色及酶活性測定結果表明TPS10啟動子的-300至-200區(qū)域為啟動子保持蟲害誘導活性的功能序列,其中的GCC-box為TPS10基因表達的一個關鍵元件。同時189份玉米自交系品種的檢測結果表明GCC-box在不同玉米自交系的TPS10啟動子中是保守的。以TPS10啟動子的-300至-200區(qū)域為誘餌序列,酵母單雜交高通量篩選擬南芥轉錄因子文庫獲得7個擬南芥AP2/ERF轉錄因子。通過系統(tǒng)進化樹分析得到與7個擬南芥AP2/ERF轉錄因子進化關系較近的17個玉米ERF轉錄因子。qPCR結果表明17個玉米ERF轉錄因子中只有EREB58受蟲害和MeJA誘導表達,并且它的表達模式和TPS10的非常類似。亞細胞定位結果顯示EREB58特異定位在細胞核中。酵母單雜交實驗,凝膠阻滯實驗結果表明EREB58能夠與TPS10啟動子中的GCC-box特異結合。擬南芥原生質體瞬時表達分析結果表明EREB58通過與GCC-box的結合激活基因的表達,具有轉錄激活活性。轉基因玉米中過表達EREB58能夠激活TPS10基因的轉錄及其倍半萜化合物(E)-β-farnesene和(E)-a-bergamotene的釋放。相比之下,將EREB58通過RNAi敲除掉后在MeJA處理條件下TPS10基因不轉錄也無倍半萜化合物的釋放。這表明在玉米中EREB58對于TPS10的誘導表達以及倍半萜揮發(fā)物的合成都是充分必要的。此外,我們對玉米萜類合成酶基因TPS6的啟動子也進行了初步的研究,研究結果表明TPS6啟動子的-100至-1區(qū)段具有啟動子活性,下一步我們將對該區(qū)段進行詳細研究。以上實驗結果表明,玉米中EREB58通過與TPS10啟動子中的順式作用元件GCC-box結合,激活TPS10基因的表達及其倍半萜化合物的釋放,闡明了玉米萜類合成酶基因TPS10的表達調控機制,對于解析玉米防御體系的分子調控網(wǎng)絡及培育抗蟲玉米品種具有重要意義,同時也為其他植物的防御體系的分子調控網(wǎng)絡的研究提供新的思路。
[Abstract]:Zea mays L.As an important food crop and industrial raw material, it occupies an important position in the national economy.Pest is one of the key factors restricting maize yield.Previous studies have shown that terpene synthase plays an important role in indirect defense of maize.When maize is damaged by Lepidoptera larvae, the leaves release a series of terpenoids to attract the natural enemies of pests.Although maize has become a model system for the study of plant indirect defense in the past two decades, the regulation of terpene synthase gene expression has not been reported in maize.In this study, the TPS10 promoter of maize terpenoid synthase gene was used as the main research object. The aim of this study was to elucidate the regulation mechanism of TPS10 expression of terpene synthase gene in maize. The results showed that TPS10 gene was induced by insect pests and MeJA.The expression site was located in the leaves and its expression level was affected by photosynthesis to some extent. PLACE prediction results showed that there were many cis-acting elements related to plant defense in the 1727 BP TPS10 promoter sequence, such as GCC-boxG-box W-box.The results of GUS histochemical staining and enzyme activity determination of transgenic Arabidopsis thaliana showed that the regions of -300 to -200 of the TPS10 promoter were the functional sequences of the promoter to maintain the insect-inducing activity, and the GCC-box was a key element of TPS10 gene expression.The results showed that GCC-box was conserved in TPS10 promoter of different maize inbred lines.Using the -300 to -200 regions of TPS10 promoter as bait sequences, seven Arabidopsis AP2/ERF transcription factors were obtained by yeast single hybrid high-throughput screening of Arabidopsis transcription factor library.Through phylogenetic tree analysis, 17 maize ERF transcription factors, which were closely related to the evolution of 7 AP2/ERF transcription factors in Arabidopsis thaliana, were obtained. The results showed that only EREB58 was induced by insect pests and MeJA among the 17 ERF transcription factors.And its expression pattern is very similar to that of TPS10.Subcellular localization showed that EREB58 was specifically located in the nucleus.The results of yeast single hybridization and gel block assay showed that EREB58 could specifically bind to GCC-box in TPS10 promoter.Transient expression analysis of Arabidopsis protoplasts showed that EREB58 had transcriptional activation activity by binding to GCC-box.Overexpression of EREB58 in transgenic maize can activate the transcription of TPS10 gene and the release of sesquiterpene compounds such as 尾 -farnesene and Ela-a-bergamotene.In contrast, the TPS10 gene was not transcribed and no sesquiterpene compounds were released under MeJA treatment after EREB58 was knocked out by RNAi.This indicated that EREB58 was necessary for the induction of TPS10 expression and the synthesis of sesquiterpene volatiles in maize.In addition, we also studied the promoter of maize terpene synthase gene TPS6. The results showed that the -100 to -1 region of TPS6 promoter had promoter activity.The results show that EREB58 activates the expression of TPS10 gene and the release of sesquiterpene compounds by binding to GCC-box, a cis-acting element in TPS10 promoter, and elucidates the mechanism of TPS10 expression in maize.It is of great significance to analyze the molecular regulatory network of maize defense system and to develop insect-resistant maize varieties. At the same time, it also provides a new idea for the study of molecular control network of other plant defense systems.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S513

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