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(nóng)業(yè)大學(xué)》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(jīng)濟中占據(jù)重要地位。蟲害是制約玉米產(chǎn)量的關(guān)鍵因素之一。已有的研究結(jié)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(jīng)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(diào)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(diào)控機制。qPCR結(jié)果表明TPS10基因受蟲害和MeJA誘導(dǎo)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(yù)測結(jié)果顯示1727 bp的TPS10啟動子序列中存在多個與植物防御相關(guān)的順式作用元件,如GCC-box,G-box,W-box。轉(zhuǎn)基因擬南芥的GUS組織化學(xué)染色及酶活性測定結(jié)果表明TPS10啟動子的-300至-200區(qū)域為啟動子保持蟲害誘導(dǎo)活性的功能序列,其中的GCC-box為TPS10基因表達的一個關(guān)鍵元件。同時189份玉米自交系品種的檢測結(jié)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(zhuǎn)錄因子文庫獲得7個擬南芥AP2/ERF轉(zhuǎn)錄因子。通過系統(tǒng)進化樹分析得到與7個擬南芥AP2/ERF轉(zhuǎn)錄因子進化關(guān)系較近的17個玉米ERF轉(zhuǎn)錄因子。qPCR結(jié)果表明17個玉米ERF轉(zhuǎn)錄因子中只有EREB58受蟲害和MeJA誘導(dǎo)表達,并且它的表達模式和TPS10的非常類似。亞細(xì)胞定位結(jié)果顯示EREB58特異定位在細(xì)胞核中。酵母單雜交實驗,凝膠阻滯實驗結(jié)果表明EREB58能夠與TPS10啟動子中的GCC-box特異結(jié)合。擬南芥原生質(zhì)體瞬時表達分析結(jié)果表明EREB58通過與GCC-box的結(jié)合激活基因的表達,具有轉(zhuǎn)錄激活活性。轉(zhuǎn)基因玉米中過表達EREB58能夠激活TPS10基因的轉(zhuǎn)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(zhuǎn)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(dǎo)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(jié)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(xì)研究。以上實驗結(jié)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(jié)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(diào)控機制,對于解析玉米防御體系的分子調(diào)控網(wǎng)絡(luò)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(diào)控網(wǎng)絡(luò)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(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S513

【共引文獻】

相關(guān)期刊論文 前10條

1 李姍姍;王明力;吳映梅;;外源信號分子在果疏保鮮中的應(yīng)用[J];貴州農(nóng)業(yè)科學(xué);2013年08期

2 陸雯;潘璐琪;王雪艷;;水楊酸及茉莉酸介導(dǎo)植物抗病性的研究進展[J];貴州農(nóng)業(yè)科學(xué);2013年10期

3 付強;鄒頡;趙杰宏;王軼;任學(xué)良;;植物氧化還原蛋白質(zhì)組學(xué)的研究進展[J];貴州農(nóng)業(yè)科學(xué);2013年12期

4 倪祥銀;齊澤民;廖姝;段輝國;;外源水楊酸對NaCl脅迫下大豆種子萌發(fā)和幼苗生長生理的影響[J];西北植物學(xué)報;2014年01期

5 馬勇;莘少紅;巴德仁貴;哈斯阿古拉;;甜瓜EIN3/EILs基因家族全基因組鑒定與進化分析[J];基因組學(xué)與應(yīng)用生物學(xué);2014年01期

6 任夢云;馮仁軍;史后蕊;云天艷;張恒;王靜毅;盧利方;張銀東;;木薯乙烯信號途徑重要基因的生物信息學(xué)及表達分析[J];分子植物育種;2014年03期

7 夏方山;毛培勝;閆慧芳;王明亞;;水楊酸對植物種子及幼苗抗逆性的影響[J];草業(yè)科學(xué);2014年07期

8 王春麗;海江波;田建華;楊建利;趙曉光;;油菜終花后角果和葉片光合對籽粒產(chǎn)量和品質(zhì)的影響[J];西北植物學(xué)報;2014年08期

9 何樂;肖牧;徐健;阮穎;;酵母雙雜交表達載體pGADT7-AtTGA2的構(gòu)建及酵母菌的轉(zhuǎn)化[J];安徽農(nóng)業(yè)科學(xué);2014年30期

10 王海波;鄒竹榮;龔明;;基于RNA-Seq數(shù)據(jù)篩選的小桐子抗冷相關(guān)基因的表達模式及其聚類分析[J];基因組學(xué)與應(yīng)用生物學(xué);2014年06期

相關(guān)博士學(xué)位論文 前10條

1 李冉;水稻抗蟲相關(guān)基因OsWRKY24、OsWRKY70和OsNPR1的功能解析[D];浙江大學(xué);2012年

2 鄭冬超;調(diào)節(jié)轉(zhuǎn)運蛋白AtNRT1.1、AtNRT2.1及PdAMT1.1基因表達對擬南芥和楊樹氮素吸收的影響研究[D];北京林業(yè)大學(xué);2013年

3 藍虹霞;兩個水稻泛素化相關(guān)鋅指蛋白及鋅轉(zhuǎn)運體蛋白OZT1的功能研究[D];南京農(nóng)業(yè)大學(xué);2012年

4 高峰;甜瓜果實發(fā)育相關(guān)基因的鑒定、表達特性及功能分析[D];內(nèi)蒙古大學(xué);2013年

5 陳全助;福建桉樹焦枯病菌鑒定及其誘導(dǎo)下桉樹轉(zhuǎn)錄組和蛋白組學(xué)研究[D];福建農(nóng)林大學(xué);2013年

6 蔡洪生;小麥TaMYB3R1抗逆功能與轉(zhuǎn)Hpal_(10-42)小麥對赤霉病和蚜蟲抗性的初步研究[D];南京農(nóng)業(yè)大學(xué);2011年

7 李寶燕;煙草WD40蛋白TTG2對生長發(fā)育和抗病性的調(diào)控作用[D];南京農(nóng)業(yè)大學(xué);2012年

8 張振華;生防多粘芽孢桿菌SQR-21的定殖與誘導(dǎo)植物系統(tǒng)抗性研究[D];南京農(nóng)業(yè)大學(xué);2011年

9 聶加寧;磷脂酸與MPK6和MKK9在擬南芥響應(yīng)鹽脅迫中的相互作用[D];南京農(nóng)業(yè)大學(xué);2012年

10 余義和;中國野生華東葡萄泛素連接酶基因抗白粉病功能研究[D];西北農(nóng)林科技大學(xué);2013年

，

本文編號：1722674